ABCMdb

ABCC7 p.Lys464Ala

ClinVar:	c.1392G>T , p.Lys464Asn ? , not provided
Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), L: D (95%), M: D (95%), N: D (91%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

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98 Two mutations, K464A (NBD1) and K1250A (NBD2) reduced ATP binding and hydrolysis [60-64]. Login to comment
100 In contrast, K464A led singly to a reduced overall hydrolytic activity [61,62,65]. Login to comment
103 Indeed, K1250A dramatically prolonged burst duration, suggesting that hydrolysis at NBD2 might be coupled to burst termination [52,65,67], whereas K464A slowed channel opening to a burst, suggesting that NBD1 might be a site of ATP interactions governing opening [68]. Login to comment

[hide] CFTR channel gating: incremental progress in irrev... J Gen Physiol. 1999 Jul;114(1):49-53. Csanady L, Gadsby DC
CFTR channel gating: incremental progress in irreversible steps.
J Gen Physiol. 1999 Jul;114(1):49-53., [PMID:10398691]

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13 The CFTR mutants K464A and K1250A, for instance, lie at the heart of challenges to the simple answers to both key questions. Login to comment
15 And recent direct measurements on purified, reconstituted CFTR have revealed virtual abolition of ATPase activity by K1250A, a more than sevenfold reduction of ATP hydrolysis (compared with WT) for K464A, but only an approximately twofold decrement in open probability (Po) for K1250A channels (because the effect of their markedly slower closing is more than offset by that of their slowed opening) and an even smaller drop in Po (due to slightly slower opening) for K464A relative to WT (Ramjeesingh et al., 1999), prompting the conclusion that ATP hydrolysis and channel gating are not tightly coupled. Login to comment
17 Although it is harder to reconcile the substantially reduced ATPase activity of K464A with its barely altered gating (Ramjeesingh et al., 1999), others have noted that K464A CFTR channels open two- (Gunderson and Kopito, 1995) or fivefold (Carson et al., 1995) more slowly than WT. Login to comment

[hide] Dual effects of ADP and adenylylimidodiphosphate o... J Gen Physiol. 1999 Jul;114(1):55-70. Weinreich F, Riordan JR, Nagel G
Dual effects of ADP and adenylylimidodiphosphate on CFTR channel kinetics show binding to two different nucleotide binding sites.
J Gen Physiol. 1999 Jul;114(1):55-70., [PMID:10398692]

Abstract [show]
The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl)ethyl]ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay, whereas ATP-P3-[1-(2-nitrophenyl)ethyl]ester only decelerated the conductance increase upon ATP addition. ADP decelerated the conductance increase upon ATP addition and accelerated the conductance decay upon ATP withdrawal. The results present the first direct evidence that AMP-PNP binds to two sites on the CFTR. The effects of ADP also suggest two different binding sites because of the two different modes of inhibition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either reflect binding to NBD-A (i.e., product inhibition in the hydrolysis cycle) or allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NBD-A.

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324 Their conclusions were derived from studying the gating (temperature not specified) and the ATPase activity (at 30ЊC) of purified CFTR protein, either WT or the mutants K464A or K1250A. Login to comment

[hide] Regulation of CFTR Cl- channel gating by ATP bindi... Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8675-80. Ikuma M, Welsh MJ
Regulation of CFTR Cl- channel gating by ATP binding and hydrolysis.
Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8675-80., 2000-07-18 [PMID:10880569]

Abstract [show]
Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg(2+) prevents hydrolysis. We found that even in the absence of Mg(2+), ATP could support channel activity, albeit at a reduced level compared with the presence of Mg(2+). Application of ATP with a divalent cation, including the poorly hydrolyzed CaATP complex, increased the rate of opening. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg(2+) further enhanced ATP-dependent opening. These data suggest that ATP alone can open the channel and that divalent cations increase ATP binding. Consistent with this conclusion, when we mutated an aspartate thought to bind Mg(2+), divalent cations failed to increase activity compared with ATP alone. Two observations suggested that divalent cations also stabilize the open state. In wild-type CFTR, CaATP generated a long duration open state, whereas ATP alone did not. With a CFTR variant in which hydrolysis was disrupted, MgATP, but not ATP alone, produced long openings. These results suggest a gating cycle for CFTR in which ATP binding opens the channel and either hydrolysis or dissociation leads to channel closure. In addition, the data suggest that ATP binding and hydrolysis by either NBD can gate the channel.

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36 In CFTR, the NBD1 mutation K464A reduces ATPase activity to Ϸ15%, and the NBD2 mutation K1250A eliminates ATPase activity (24). Login to comment
136 We tested variants with mutations in the Walker A lysine, CFTR-K464A and -K1250A. Login to comment
143 In contrast, with CFTR-K464A, the burst duration was the same with MgATP and ATP alone (Fig. 3 B and C). Login to comment
144 There are two potential explanations for the difference between K464A and K1250A. Login to comment
146 Alternatively, in K464A, most of the gating may be due to ATP interactions with NBD2. Login to comment
155 Therefore, we studied CFTR-K1250A and CFTR-K464A at two different ATP concentrations. Login to comment
161 Yet, prolonged burst durations have not been observed with K464A (10, 11, 14, 24). Login to comment
164 However, if NBD1 has a higher ATP affinity than NBD2, this premise predicts that K464A would have long bursts at low MgATP concentrations. Login to comment
166 With 1 mM ATP and 4 mM Mg2ϩ , CFTR-K464A showed durations approximately the same as observed with wild-type CFTR. Login to comment
198 Our finding that the K464A mutation can generate bursts with a prolonged duration (Fig. 4) supports this conclusion. Login to comment
203 Effect of MgATP and ATP alone on CFTR-K1250A and -K464A. Login to comment
238 Effect of ATP concentration on gating of CFTR-K1250A (A and C) and K464A (B and D) channels. Login to comment

[hide] Differential interactions of nucleotides at the tw... J Biol Chem. 2001 Apr 20;276(16):12918-23. Epub 2001 Jan 29. Aleksandrov L, Mengos A, Chang X, Aleksandrov A, Riordan JR
Differential interactions of nucleotides at the two nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2001 Apr 20;276(16):12918-23. Epub 2001 Jan 29., 2001-04-20 [PMID:11279083]

Abstract [show]
After phosphorylation by protein kinase A, gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by the interaction of ATP with its nucleotide binding domains (NBDs). Models of this gating regulation have proposed that ATP hydrolysis at NBD1 and NBD2 may drive channel opening and closing, respectively (reviewed in Nagel, G. (1999) Biochim. Biophys. Acta 1461, 263-274). However, as yet there has been little biochemical confirmation of the predictions of these models. We have employed photoaffinity labeling with 8-azido-ATP, which supports channel gating as effectively as ATP to evaluate interactions with each NBD in intact membrane-bound CFTR. Mutagenesis of Walker A lysine residues crucial for azido-ATP hydrolysis to generate the azido-ADP that is trapped by vanadate indicated a greater role of NBD1 than NBD2. Separation of the domains by limited trypsin digestion and enrichment by immunoprecipitation confirmed greater and more stable nucleotide trapping at NBD1. This asymmetry of the two domains in interactions with nucleotides was reflected most emphatically in the response to the nonhydrolyzable ATP analogue, 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), which in the gating models was proposed to bind with high affinity to NBD2 causing inhibition of ATP hydrolysis there postulated to drive channel closing. Instead we found a strong competitive inhibition of nucleotide hydrolysis and trapping at NBD1 and a simultaneous enhancement at NBD2. This argues strongly that AMP-PNP does not inhibit ATP hydrolysis at NBD2 and thereby questions the relevance of hydrolysis at that domain to channel closing.

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23 EXPERIMENTAL PROCEDURES Materials- BHK-21 cells stably expressing wild-type human CFTR were prepared and maintained as described previously (20) as were cells expressing the K464A and K1250A mutants. Login to comment
63 A, membranes from BHK cells expressing wild-type and the K1250A (15 ␮g of protein) and K464A variants (60 ␮g of protein) were incubated with 20 ␮M 8-azido-[␣-32 P]ATP in the presence of 0.5 mM orthovanadate and processed as under "Experimental Procedures." Login to comment
65 Note that 4 times more K464A membrane protein was used than wild type and K1250. Login to comment
66 The cells expressing K464A had also been grown at 26 °C to promote maturation of the mutant CFTR. B, 15 ␮g of BHK membrane protein containing wild-type CFTR was incubated at 0 °C for 1 h with 50 ␮g/ml purified immunoglobulin from each of the mouse monoclonal antibodies. Login to comment
70 In contrast the comparable substitution in NBD1 (K464A) greatly diminished labeling compared with wild type indicating that the majority of labeling and trapping may occur at NBD1. Login to comment

[hide] ATP hydrolysis-coupled gating of CFTR chloride cha... Biochemistry. 2001 May 15;40(19):5579-86. Zou X, Hwang TC
ATP hydrolysis-coupled gating of CFTR chloride channels: structure and function.
Biochemistry. 2001 May 15;40(19):5579-86., 2001-05-15 [PMID:11341822]

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158 Although earlier studies showed that converting lysine 464 to alanine (K464A) slowed opening (31, 32), a more recent report found that single-channel gating kinetics of the K464A mutant was almost indistinguishable from that of the wild-type CFTR (46-48). Login to comment
159 The apparent affinity for ATP is little changed for the K464A channel (48). Login to comment
160 Nevertheless, the ATP hydrolysis rate of the K464A mutant is decreased (46). Login to comment
236 Their model is based mainly on the studies of the K464A and K1250A mutants, assuming mutations of the Walker A lysines diminish the level of ATP hydrolysis at respective NBDs. Login to comment
240 This model explains the results showing that the K464A mutant shows a longer open time at micromolar ATP concentrations than that at millimolar ATP concentrations, whereas the K1250A mutant shows an opposite pattern of gating in response to changes in the ATP concentration (cf. ref 38). Login to comment
242 For example, on the basis of the assumption of two open states in the NBD1 cycle, it is desirable to observe two distinct populations of open time for the K464A mutant especially at millimolar ATP concentrations. Login to comment
248 To explain this result for the K464A mutant, one needs to speculate that hydrolysis of one ATP molecule may trigger several open-close events. Login to comment

[hide] Mutations that change the position of the putative... J Biol Chem. 2002 Jan 18;277(3):2125-31. Berger AL, Ikuma M, Hunt JF, Thomas PJ, Welsh MJ
Mutations that change the position of the putative gamma-phosphate linker in the nucleotide binding domains of CFTR alter channel gating.
J Biol Chem. 2002 Jan 18;277(3):2125-31., 2002-01-18 [PMID:11788611]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is an ATP-binding cassette transporter that contains conserved nucleotide-binding domains (NBDs). In CFTR, the NBDs bind and hydrolyze ATP to open and close the channel. Crystal structures of related NBDs suggest a structural model with an important signaling role for a gamma-phosphate linker peptide that couples bound nucleotide to movement of an alpha-helical subdomain. We mutated two residues in CFTR that the structural model predicts will uncouple effects of nucleotide binding from movement of the alpha-helical subdomain. These residues are Gln-493 and Gln-1291, which may directly connect the ATP gamma-phosphate to the gamma-phosphate linker, and residues Asn-505 and Asn-1303, which may form hydrogen bonds that stabilize the linker. In NBD1, Q493A reduced the frequency of channel opening, suggesting a role for this residue in coupling ATP binding to channel opening. In contrast, N505C increased the frequency of channel opening, consistent with a role for Asn-505 in stabilizing the inactive state of the NBD. In NBD2, Q1291A decreased the effects of pyrophosphate without altering other functions. Mutations of Asn-1303 decreased the rate of channel opening and closing, suggesting an important role for NBD2 in controlling channel burst duration. These findings are consistent with both the bacterial NBD structural model and gating models for CFTR. Our results extend models of nucleotide-induced structural changes from bacterial NBDs to a functional mammalian ATP-binding cassette transporter.

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22 However, the effects of mutations in the two NBDs are not symmetrical; the K1250A mutation dramatically prolongs the burst duration, whereas the K464A mutation reduces the frequency of channel opening but does not change burst duration. Login to comment

[hide] The First Nucleotide Binding Domain of Cystic Fibr... J Biol Chem. 2002 May 3;277(18):15419-25. Epub 2002 Feb 22. Aleksandrov L, Aleksandrov AA, Chang XB, Riordan JR
The First Nucleotide Binding Domain of Cystic Fibrosis Transmembrane Conductance Regulator Is a Site of Stable Nucleotide Interaction, whereas the Second Is a Site of Rapid Turnover.
J Biol Chem. 2002 May 3;277(18):15419-25. Epub 2002 Feb 22., 2002-05-03 [PMID:11861646]

Abstract [show]
As in other adenine nucleotide binding cassette (ABC) proteins the nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator (CFTR) bind and hydrolyze ATP and in some manner regulate CFTR ion channel gating. Unlike some other ABC proteins, however, there are preliminary indications that the two domains of CFTR are nonequivalent in their nucleotide interactions (Szabo, K., Szakacs, G., Hegeds, T., and Sarkadi, B. (1999) J. Biol. Chem. 274, 12209-12212; Aleksandrov, L., Mengos, A., Chang, X., Aleksandrov, A., and Riordan, J. R. (2001) J. Biol. Chem. 276, 12918-12923). We have now characterized the interactions of the 8-azido-photoactive analogues of ATP, ADP, and 5'-adenyl-beta,gamma-imidodiphosphate (AMP-PNP) with the two domains of functional membrane-bound CFTR. The results show that the two domains appear to act independently in the binding and hydrolysis of 8-azido-ATP. At NBD1 binding does not require a divalent cation. This binding is followed by minimal Mg(2+)-dependent hydrolysis and retention of the hydrolysis product, 8-azido-ADP, but not as a vanadate stabilized post-hydrolysis transition state complex. In contrast, at NBD2, MgN(3)ATP is hydrolyzed as rapidly as it is bound and the nucleoside diphosphate hydrolysis product dissociates immediately. Confirming this characterization of NBD1 as a site of more stable nucleotide interaction and NBD2 as a site of fast turnover, the non-hydrolyzable N(3)AMP-PNP bound preferentially to NBD1. This demonstration of NBD2 as the rapid nucleotide turnover site is consistent with the strong effect on channel gating kinetics of inactivation of this domain by mutagenesis.

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28 To distinguish between these possibilities, the Walker A lysine mutants K464A and K1250A were used; K464A ablated labeling of NBD1 without influencing that at NBD2, and hence the N3ADP that labeled * This work was supported by Grant DK51619 from the NIDDK, National Institutes of Health and by the Cystic Fibrosis Foundation. Login to comment
43 Stable BHK-21 cell lines expressing wild-type and K464A and K1250A variants of CFTR were established and cultured as described previously (16, 17). Login to comment
130 As expected, the mutations K464A and K1250A prevented photolabeling with either 8-azido-ATP or 8-azido-ADP of NBD1 and NBD2, respectively. Login to comment
131 However, there was no indication that the mutation in one domain had any influence on the labeling of the other, i.e. in K464A, NBD2 was labeled as in wild-type and in K1250A, NBD1 was not different from wild type. Login to comment
141 Hence, as with a mutation (K464A) that prevents labeling of NBD1 by N3ATP, occupation of the domain by AMP-PNP leaves the labeling of NBD2 with [␣-32 P]N3ATP entirely intact. Login to comment
191 Membranes from BHK cells expressing wild-type and K464A and K1250A variants of CFTR were incubated as in Figs. Login to comment
193 The mobility of the large 95-kDa, NBD2 band is increased in the K464A variant, which does not mature and completely acquire the complex oligosaccharide chains, which contribute to the apparent size of the wild-type band. Login to comment

[hide] Mutation of Walker-A lysine 464 in cystic fibrosis... J Physiol. 2002 Mar 1;539(Pt 2):333-46. Powe AC Jr, Al-Nakkash L, Li M, Hwang TC
Mutation of Walker-A lysine 464 in cystic fibrosis transmembrane conductance regulator reveals functional interaction between its nucleotide-binding domains.
J Physiol. 2002 Mar 1;539(Pt 2):333-46., 2002-03-01 [PMID:11882668]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel bears two nucleotide-binding domains (NBD1 and NBD2) that control its ATP-dependent gating. Exactly how these NBDs control gating is controversial. To address this issue, we examined channels with a Walker-A lysine mutation in NBD1 (K464A) using the patch clamp technique. K464A mutants have an ATP dependence (EC(50) approximate 60 microM) and opening rate at 2.75 mM ATP (approximately 2.1 s(-1)) similar to wild type (EC(50) approximate 97 microM; approximately 2.0 s(-1)). However, K464A's closing rate at 2.75 mM ATP (approximately 3.6 s(-1)) is faster than that of wild type (approximately 2.1 s(-1)), suggesting involvement of NBD1 in nucleotide-dependent closing. Delay of closing in wild type by adenylyl imidodiphosphate (AMP-PNP), a non-hydrolysable ATP analogue, is markedly diminished in K464A mutants due to reduction in AMP-PNP's apparent on-rate and acceleration of its apparent off-rate (approximately 2- and approximately 10-fold, respectively). Since the delay of closing by AMP-PNP is thought to occur via NBD2, K464A's effect on the NBD2 mutant K1250A was examined. In sharp contrast to K464A, K1250A single mutants exhibit reduced opening (approximately 0.055 s(-1)) and closing (approximately 0.006 s(-1)) rates at millimolar [ATP], suggesting a role for K1250 in both opening and closing. At millimolar [ATP], K464A-K1250A double mutants close approximately 5-fold faster (approximately 0.029 s(-1)) than K1250A but open with a similar rate (approximately 0.059 s(-1)), indicating an effect of K464A on NBD2 function. In summary, our results reveal that both of CFTR's functionally asymmetric NBDs participate in nucleotide-dependent closing, which provides important constraints for NBD-mediated gating models.

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12 To address this issue, we examined channels with a Walker-A lysine mutation in NBD1 (K464A) using the patch clamp technique. Login to comment
13 K464A mutants have an ATP dependence (EC50 ∆ 60 m) and opening rate at 2.75 m ATP (~2.1 s_1 ) similar to wild type (EC50 ∆ 97 m; ~2.0 s_1 ). Login to comment
15 Delay of closing in wild type by adenylyl imidodiphosphate (AMP-PNP), a non-hydrolysable ATP analogue, is markedly diminished in K464A mutants due to reduction in AMP-PNP`s apparent on-rate and acceleration of its apparent off-rate (~2and ~10-fold, respectively). Login to comment
17 In sharp contrast to K464A, K1250A single mutants exhibit reduced opening (~0.055 s_1 ) and closing (~0.006 s_1 ) rates at millimolar [ATP], suggesting a role for K1250 in both opening and closing. Login to comment
18 At millimolar [ATP], K464A-K1250A double mutants close ~5-fold faster (~0.029s_1 )thanK1250Abutopenwithasimilarrate(~0.059s_1 ),indicatinganeffectofK464Aon NBD2 function. Login to comment
24 Supporting this idea, some investigators reported that a mutation in NBD1`s Walker-A motif (i.e. K464A) reduces CFTR`s opening rate (Carson et al. 1995; Gunderson & Kopito, 1995; Vergani et al. 2000). Login to comment
25 However, those earlier reports are being challenged by more recent demonstrations that K464A has little effect on channel opening (Sugita et al. 1998; Ramjeesingh et al. 1999; present study). Login to comment
29 To examine the contribution of NBD1 during CFTR gating, we assessed the kinetic properties of the Walker-A mutant K464A. Login to comment
31 The plasmids K464A-pRBG4 and K1250A-pRBG4 were gifts from Dr R. R. Kopito (Stanford University, CA, USA), and the plasmid CFTRwt-pBQ4.7 and the retroviral vector pLJ were gifts from Dr M. Drumm (Case Western Reserve University, Cleveland, OH, USA). Login to comment
32 To construct K464A-pBQ and K1250A-pBQ, the 0.7 kb BspEI-BstZ171 fragment from K464A-pRBG4 and the 3.0 kb BspEI-NcoI fragment from K1250A-pRBG4, respectively, replaced the corresponding ones in CFTRwt-pBQ4.7. Login to comment
34 For construction of K464A-pLJ, the 3.6 kb BspEI-SalI fragment from K464A-pBQ was exchanged with the corresponding one in WT-pLJ. Login to comment
35 To generate WT-pCDNA, K464A-pCDNA and K1250A-pCDNA,the4.7 kbPstIfragmentsfromthecorresponding pBQ constructs were subcloned into the PstI site of pCDNA. Login to comment
36 For the creation of K464A-K1250A-pCDNA, the 2.7 kb BspEI-PflMI fragment from K464A-pCDNA was used to substitute the corresponding region in K1250A-pCDNA. Login to comment
40 To obtain recordings with few channels per patch, we used NIH3T3 cells stably transfected with wild type CFTR (Berger et al. 1991), CFTR-K1250A and CFTR-K464A (Zeltwanger et al. 1999; present study). Login to comment
54 Macroscopicdose-responseanalysisofK464Achannels To quantify the ATP dependence of K464A channels, we normalized baseline-subtracted mean current levels elicited by test [ATP] against mean current levels at 2.75 m ATP as previously described (Zeltwanger et al. 1999; see Fig. 1). Login to comment
60 An analysis of flickery events in K464A channels using 100 Hz filtering and 500 Hz sampling justified the 50 ms cut-off (data not shown). Login to comment
68 However, since the solution exchange in our system is relatively slow (~5 s; cf. Zeltwanger et al. 1999), relaxation times for K464A, but not wild type, are probably rate limited more by nucleotide removal rather than actual channel closing (see Results). Login to comment
69 To estimate more accurately the AMP-PNP-dependent open time for K464A channels, we analysed recordings with only one or two channels; open events where two channels were open simultaneously were averaged and included as described previously (Fenwicketal.1982;Wangetal.1998).Dwelltimeswererankedin order of decreasing duration, normalized by the number of events and displayed as survivor plots (i.e. the probability of still being open at time t given that the channel was open at time zero versus time). Login to comment
72 Based on our open time analysis of K464A channels in the presence of 250 µ ATP and 1 m AMP-PNP (see above), we used a cut-off of 842 ms for both wild type and K464A channels. Login to comment
73 Use of this cut-off for both types of channels is justifiable, since both K464A and wild type have similar open times (i.e. dwell times in O) at 250 µ ATP alone. Login to comment
75 EstimationofkineticparametersforK1250Aand K464A-K1250A For recordings of quasi-macroscopic K1250A and K464A-K1250A channel currents, open probability was estimated by means of variance analysis (Sigworth, 1980): Po = (1 _ (s2 /Ii)), where Po represents open probability, s2 the variance of steady-state current, I the mean steady-state current and i the single channel amplitude. Login to comment
80 RESULTS As a step towards understanding how CFTR`s NBDs participate in gating, we examined the kinetic behaviour of the NBD1 mutant K464A, the NBD2 mutant K1250A and double mutant K464A-K1250A. Login to comment
81 K464A mutants were examined first. Login to comment
82 To determine whether K464A affected CFTR`s ATP dependence, we performed a dose-response analysis. Login to comment
83 Some earlier reports indicated that K464A reduced ATP affinity (Anderson & Welsh, 1992; Vergani et al. 2000). Login to comment
89 From the fit, the Km for K464A channels was 59 ± 9 µ. Login to comment
91 This comparison indicates that the K464A mutation had little effect on ATP sensitivity. Login to comment
92 To estimate the ATP dependence of open probability in K464A mutants, the Po was measured in patches with one to four channels. Login to comment
97 The fit results closely matched the measured data; the measured mean Po for K464A channels was 0.37 ± 0.02 (n = 18) at 2.75 m ATP, 0.21 ± 0.05 (n = 5) at 100 µ ATP, and 0.19 ± 0.02 (n = 3) at 50 µ ATP, indicating that the EC50 for ATP lies between 50 and 100 µ. Login to comment
100 Furthermore, a comparison of K464A to wild type (Zeltwanger et al. 1999; present study, Fig. 1B) shows little change in ATP sensitivity of Po. Login to comment
101 A Michaelis-Menten fit to our previous wild type data gave a Km of 137 µ with a maximum Po of 0.41, slightly higher than K464A. Login to comment
102 We next tested how [ATP] affected channel opening and closing rates in K464A mutants. Login to comment
105 ATP dose-response relationships for CFTR-K464A A, representative trace of macroscopic CFTR-K464A channel current stimulated by 2.75 m and 25 µ ATP after steady-state activation by PKA phosphorylation (not shown). Login to comment
106 B, trace of CFTR-K464A channels exposed to different [ATP] after steady-state activation by PKA and ATP. Login to comment
107 C, macroscopic dose-response relationship for CFTR-K464A (•; present study) and wild type CFTR (ª; data taken from Zeltwanger et al. 1999). Login to comment
108 D, open probability versus [ATP] for CFTR-K464A (0; present study) and wild type (1; data taken from Zeltwanger et al. 1999). Login to comment
109 The maximum Po for CFTR-K464A at 2.75 m ATP is ~0.37. Login to comment
112 Dashed lines are fits of the Michaelis-Menten equation to the CFTR-K464A data (see Methods). Login to comment
113 shows sweeps from a recording of a single CFTR-K464A channel exposed to 2.75 m and 100 µ ATP. Login to comment
115 In 100 µ ATP the K464A channel exhibits longer closures, while the duration of openings at both concentrations appears similar. Login to comment
116 Figure 2B shows the closed and open time distributions from the K464A recordings in Fig. 2A. Login to comment
121 The relationship between rates and [ATP] for K464A mutants is shown in Fig. 3A; the data shown represent the mean rates pooled from 18 experiments. Login to comment
124 To quantify the ATP sensitivity of opening in K464A mutants, the data were fitted to a Michaelis-Menten equation (Fig. 3A). Login to comment
127 Our findings confirm that ATP-dependent opening is not impaired by the K464A mutation, qualitatively consistent with the data of Sugita et al. (1998) and Ramjeesingh et al. (1999). Login to comment
128 Unlike opening, K464A channel closing exhibits little, if any, dependence on ATP. Login to comment
131 Single-channel kinetics of CFTR-K464A A, representative sweeps from experiments with single CFTR-wild type (left-hand sweeps; taken from Zeltwanger et al. 1999) and CFTR-K464A channels (right-hand sweeps) exposed to 2.75 m (top sweeps) and 100 µ MgATP (bottom sweeps) subsequent to activation by PKA (40 U ml_1 ) and ATP (2.75 m). Login to comment
133 B, survivor plots of closed (left-hand panels) and open dwell times (right-hand panels) at 2.75 m (top panels) and 100 µ MgATP (bottom panels) for the single CFTR-K464A channel shown in A (cf. wild type distributions in Zeltwanger et al. 1999). Login to comment
135 hand, a comparison of closing rates for K464A and wild type at 2.75 m ATP does reveal a difference (3.6 ± 0.3 s_1 , n = 18 versus 2.1 ± 0.3 s_1 , n = 6, respectively; P < 0.005; Fig. 3B). Login to comment
136 Thus, K464A appears to abolish the ATP dependence of the closing rate seen in wild type. Login to comment
137 From this result, we suspected that K464A might affect the second functional site for ATP, which is presumed responsible for prolonging open time in wild type channels at millimolar [ATP] (Zeltwanger et al. 1999). Login to comment
138 Since this second functional site for ATP is thought to be the site for AMP-PNP action (Hwang et al. 1994; Carson et al. 1995; Mathews et al. 1998b; Zeltwanger et al. 1999), we examined whether K464A affected AMP-PNP`s prolongation of channel opening. Login to comment
144 Prolonged openings can also be seen in K464A channels exposed to AMP-PNP, but with lower frequency and shorter duration. Login to comment
145 For example, Fig. 4A (lower trace) shows a single K464A channel activated by PKA (40 U ml_1 ) and ATP (250 µ), then exposed to AMP-PNP (1 m). Login to comment
146 Unlike the wild type example, the K464A channel took much longer to become locked open. Login to comment
148 These two examples suggest that the K464A mutation A. C. Powe, Jr, L. Al-Nakkash, M. Li and T.-C. Hwang338 J. Physiol. 539.2 Figure 3. Login to comment
149 Dependence of CFTR-K464A closing and opening rates on [ATP] A, plot of mean opening rates (reciprocals of mean closed times; 0) and closing rates (reciprocals of mean opened times; ª) versus [ATP] for CFTR-K464A. Login to comment
151 B, comparison of opening and closing rates for CFTR-K464A and wild type at 2.75 m ATP. Login to comment
152 Asterisks indicate a significant difference between closing rates for wild type and K464A (P < 0.005). Login to comment
154 AMP-PNP weakly locks open CFTR-K464A A, representative sweeps from experiments with wild type (top trace) and CFTR-K464A single channels (bottom trace) exposed first to PKA (40 U ml_1 ) and MgATP (250 µ), then with the addition of AMP-PNP (1 m). Login to comment
155 Arrows indicate the baseline, downward deflections channel openings. B, survivor plot of open dwell times for CFTR-K464A. Login to comment
158 reduces the ability to become locked open and to stay locked open by AMP-PNP, further substantiating the idea that K464A affects the second functional site for ATP. Login to comment
160 The opening of K464A channels is slowed by AMP-PNP as well. Login to comment
161 The mean closed time for K464A channels exposed to 250 µ ATP and 1 m AMP-PNP was 1366 ± 260 ms (n = 6), approximately threefold longer than in 250 µ ATP alone (~465 ms; Fig. 3A). Login to comment
163 To quantify the extent to which K464A affects the second functional site, we first estimated the duration of AMP-PNP-dependent locked open events using current relaxation time courses upon removal of AMP-PNP. Login to comment
165 Figure 5A shows a comparison of relaxations from wild type (top trace) and K464A channels (bottom trace). Login to comment
167 Wild type channels close slowly, while K464A channels rapidly shut, indicating a shortened locked open duration in the mutant. On average, wild type channels show a mean relaxation time constant of 105 ± 22 s (n = 5) and K464A channels 12 ± 3 s (n = 5; P < 0.01) (Fig. 5B). Login to comment
168 However, given that the solution exchange in our system is relatively slow (~5 s; cf. Zeltwanger et al. 1999), relaxation times for K464A are probably rate limited more by nucleotide removal than actual channel closing. Login to comment
170 To obtain a more accurate estimate for locked open event duration in K464A channels, we analysed the open time distribution from patches with one or two channels in the presence of PKA, ATP and AMP-PNP (Fig. 4B; see Methods). Login to comment
172 One component was ~300 ms, consistent with the mean open time for K464A channels in 250 µ ATP alone (Fig. 4B; cf. Fig. 3). Login to comment
178 In addition to reducing the time spent locked open, the K464A mutation also impairs the ability to become locked open, as exemplified by Fig. 4A. Login to comment
179 In that example, the K464A channel opens and shuts many times before being locked open, while wild type opens and shuts only a few times before locking open. Login to comment
181 K464A enhances the dissociation of AMP-PNP A, representative traces from experiments with macroscopic currents from CFTR-wild type (top trace) and CFTR-K464A channels (bottom trace) exposed to PKA (40 U ml_1 ), MgATP (250 µ) and AMP-PNP (1 m). Login to comment
183 For the traces shown, the mean relaxation time constant for wild type is 64.8 ± 0.1 s and for CFTR-K464A is 9.1 ± 0.1 s. B, mean relaxation time constants (± ...) Login to comment
184 for wild type and K464A channels. Login to comment
185 Asterisks indicate a significant difference between wild type and K464A (P < 0.01). Login to comment
190 Using the open duration data from K464A channels exposed to AMP-PNP (Fig. 4B), we calculated a cut-off of 842 ms; openings longer than 842 ms were L state and shorter ones O. Login to comment
191 This cut-off was used to classify open events for both wild type and K464A channels. Login to comment
193 Figure 6A shows the distributions obtained from two experiments, one with wild type channels and the other with K464A. Login to comment
194 In the experiments shown, the locking rate for wild type (ª) was 1110 ± 70 s_1 _1 (21 events) and for K464A (1) 370 ± 10 s_1 _1 (49 events), qualitatively consistent with the examples in Fig. 4A. Login to comment
195 On average, wild type channels exhibited a faster locking rate (890 ± 140 s_1 _1 , n = 3) than K464A (560 ± 110 s_1 _1 , n = 6; P < 0.05; Fig. 6B), indicating that the mutation impairs AMP-PNP`s ability to lock open CFTR. Login to comment
198 We wondered whether K464A reduces channel open time in K1250A mutants as it does with AMP-PNP. Login to comment
199 To test this idea, we determined the mean open time for both K1250A single mutant and K464A-K1250A double mutant channels using relaxation time courses upon ATP withdrawal (Fig. 7A). Login to comment
200 In the examples shown, the double mutant relaxes more rapidly than the single mutant. On average, K464A-K1250A channel currents decay fivefold morequicklythanK1250A(34 ± 7 s,n = 5versus167 ± 37 s, n = 6, respectively; Fig. 7C; cf. Zeltwanger et al.1999). Login to comment
202 We also examined the open probability of K1250A and K464A-K1250A. Login to comment
204 K464A reduces the apparent on-rate of AMP-PNP A, Semilog plot of a probability function determined by measuring the cumulative time channels spent in the O state before entering the L state (see Methods). Login to comment
205 The dwell times are from individual experiments for CFTR-K464A (1) and CFTR-wild type (ª). Login to comment
207 B, comparison of the mean locking rates for CFTR-wild type and CFTR-K464A. Login to comment
208 Asterisk indicates a significant difference between wild type and K464A (P < 0.05). Login to comment
210 In this example, all four K1250A channels remain open for most of the sweep, whereas only two of the three double mutants channels are open most of the time, suggesting a lower Po for K464A-K1250A. Login to comment
212 Our estimates of Po for both mutants are much higher than previously reported (~ 0.2_0.34 for K1250A and ~ 0.25 for K464A-K1250A; Carson et al. 1995; Ramjeesingh et al. 1999; but cf. ~0.9 for K1250A; Gunderson & Kopito, 1995). Login to comment
214 To see whether the reduced Po of K464A-K1250A mutants arises only from shorter open times, we calculated mean closed times from steady-state Po and relaxation time constants (see Methods). Login to comment
216 Thus, K1250A prolongs closed time >30-fold compared either to wild type or to the K464A single mutant (Fig. 3B). Login to comment
218 The mean closed time for K464A-K1250A (17 ± 4 s, n = 5; Fig. 7B) is similar to that for K1250A (P ∆ 0.42). Login to comment
219 Our results show that the lower Po in the double mutant is mostly due to shortening of K1250A`s long open time by K464A. Login to comment
221 We tested whether K464A-K1250A behaved in a similar manner. Login to comment
222 A typical example of K464A-K1250A`s gating behaviour is shown in Fig. 8A. Login to comment
227 We then examined the open time distribution of K464A-K1250A channels in the presence Functional interaction between nucleotide binding domains of CFTRJ. Physiol. 539.2 341 Figure 7. Login to comment
228 K464A shortens K1250A relaxation A, representative trace of macroscopic current relaxations from CFTR-K1250A (top trace) and CFTR-K464A-K1250A double mutant channels upon withdrawal of PKA (40 U ml_1 ) and ATP (1 m). Login to comment
229 Mean relaxation time constant for the CFTR-K1250A trace shown is 110 ± 1 s and for CFTR-K464A-K1250A is 30 ± 1 s. B, few-channel traces of CFTR-K1250A and CFTR-K464A-K1250A at the steady state in 2.75 m ATP. Login to comment
230 Dashed lines indicate baseline (all channels closed); marks at the left indicate open channel current levels (a total of 4 channels for K1250A and 3 for K464A-K1250A). Login to comment
231 C, comparison of steady-state Po, mean open (relaxation) times and mean closed times for CFTR-K1250A and CFTR-K464A-K1250A. Login to comment
232 Asterisks indicate significant differences between CFTR-K1250A and CFTR-K464A-K1250A (**P < 0.01; ***P < 0.005). Login to comment
234 A single exponential fit to the distribution provides an estimated open time of 241 ± 3 ms, similar to the open time of wild type, K464A and K1250A at 10 µ ATP (~250 ms; Zeltwanger et al. 1999; present study, Fig. 3A). Login to comment
235 Thus, K464A had little effect on brief openings seen in K1250A at micromolar [ATP]. Login to comment
241 We demonstrate that K1250A, but not K464A, affects the opening rate. Login to comment
242 We also show that both K464A and K1250A affect closing at millimolar [ATP] but in opposite ways. Login to comment
243 K464A accelerates closing whereas K1250A delays it. Login to comment
249 Ramjeesingh et al. (1999) showed that K464A only partly reduced CFTR`s ATPase activity while K1250A eliminates it altogether. Login to comment
250 Aleksandrov et al. (2001) showed that K1250A had no effect on 8-azido- [a-32 P]-ATP labelling of CFTR whereas K464A drastically reduced it. Login to comment
255 It was further hypothesized that hydrolysis is the main pathway for closing under normal conditions and that blocking hydrolysis with K464A or K1250A permits closing only through the slow unbinding pathway, resulting in prolonged openings. Login to comment
256 Based on that model, one would predict that the double mutant K464A-K1250A should exhibit long openings at all ATP concentrations, since the hydrolysis pathway at both A. C. Powe, Jr, L. Al-Nakkash, M. Li and T.-C. Hwang342 J. Physiol. 539.2 Figure 8. Login to comment
257 K464A-K1250A gating at millimolar and micromolar [ATP] A, representative sweep from experiments with CFTR-K464A-K1250A channels exposed first to 1 m and then to 10 µ MgATP. Login to comment
258 Arrow indicates the baseline, downward deflections channel openings. B, survivor plot of open dwell times for CFTR-K464A-K1250A at 10 µ MgATP. Login to comment
262 We find, however, that the mean open time for K464A-K1250A is ~250 ms at 10 µ ATP and ~30 s at 2.75 m ATP (Figs 7 and 8B). Login to comment
263 The double mutant open time at 10 µ ATP is similar to that of wild type, K464A and K1250A at the same [ATP] (Zeltwanger et al. 1999; present study). Login to comment
264 This finding, together with the dramatic differences between K464A and K1250A mutants, casts considerable doubt on the idea that the NBDs function identically. Login to comment
270 The frequency and duration of these locked open events in the presence of AMP-PNP are reduced by the NBD1 mutation K464A (Figs 4, 5 and 6). Login to comment
272 The reduction of AMP-PNP`s lock open effect by K464A then suggests that lysine 464 in NBD1 regulates nucleotide action at NBD2. Login to comment
276 Because K464A reduces the apparent on-rate (i.e. locking rate) of AMP-PNP (Fig. 6), we expected that this mutation should also impair ATP`s ability to prolong open time. Login to comment
278 Furthermore, the NBD2 mutation K1250A greatly prolongs CFTR open time ~300-fold; that prolongation is then reduced ~5-fold by addition of the NBD1 mutation K464A (Fig. 7). Login to comment
288 Furthermore, Ramjeesingh et al. (1999) showed that K464A reduces ATPase activity by ~80% and K1250A virtually eliminates it, suggesting that mutating one NBD affects the biochemical activity of the other. Login to comment
299 Channel open times at 10 µ ATP for wild type, K464A, K1250A and K464A-K1250A are all ~250 ms (Zeltwanger et al. 1999; present study, Figs 3 and 8), indicating that the NBD mutations have no effect on brief openings. Login to comment
306 On the other hand, the NBD1 mutation K464A shortens openings at millimolar [ATP] (Fig. 3). Login to comment
307 K464A also impairs AMP-PNP`s ability to lock open CFTR and the channel`s ability to remain locked open (Figs 4, 5 and 6), which implies that blocking hydrolysis at NBD1 impairs the ability to enter and remain in the longer open state. Login to comment
321 If so, why does the K464A mutation not affect the opening rate? Login to comment
322 There are two possible explanations for this observation: (1) ATP binding, but not hydrolysis at NBD1, opens the channel; or (2) NBD1`s role in channel opening is not revealed by the K464A mutation. Login to comment
323 Although biochemical studies show that K464A does impair ATPase activity in isolated NBD1 (Ko & Pedersen, 1995; King & Sorscher, 1998) as well as in the whole CFTR molecule (Ramjeesingh et al. 1999), the opening rate of K464A may not be affected if opening of CFTR only requires ATP binding. Login to comment
326 Another possibility is that NBD1 may be involved in channel opening, but the domain`s role is not revealed by the K464A mutation. Login to comment
334 A. C. Powe, Jr, L. Al-Nakkash, M. Li and T.-C. Hwang J. Physiol. 539.2 Conclusions To examine the contribution of NBD1 during CFTR gating, we assessed the kinetic properties of the Walker-A mutant K464A. Login to comment
335 We found that K464A had little effect on the apparent ATP dependence or opening rate of the channel. Login to comment
337 K464A also diminished AMP-PNP`s ability to stabilize channel open state by affecting both apparent on- and off-rates of the non-hydrolysable analogue. Login to comment
338 Finally, K464A reduces the prolongation of open time seen in the NBD2 mutant K1250A at millimolar [ATP], strongly suggesting an interaction between NBD1 and NBD2 during CFTR`s open state. Login to comment
339 Although the NBD1 mutant K464A did not affect opening, the NBD2 mutant K1250A delays opening >30-fold compared to wild type. Login to comment

[hide] Distinct Mg(2+)-dependent steps rate limit opening... J Gen Physiol. 2002 Jun;119(6):545-59. Dousmanis AG, Nairn AC, Gadsby DC
Distinct Mg(2+)-dependent steps rate limit opening and closing of a single CFTR Cl(-) channel.
J Gen Physiol. 2002 Jun;119(6):545-59., [PMID:12034762]

Abstract [show]
The roles played by ATP binding and hydrolysis in the complex mechanisms that open and close cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels remain controversial. In this work, the contributions made by ATP and Mg(2+) ions to the gating of phosphorylated cardiac CFTR channels were evaluated separately by measuring the rates of opening and closing of single channels in excised patches exposed to solutions in which [ATP] and [Mg(2+)] were varied independently. Channel opening was found to be rate-limited not by the binding of ATP alone, but by a Mg(2+)-dependent step that followed binding of both ATP and Mg(2+). Once a channel had opened, sudden withdrawal of all Mg(2+) and ATP could prevent it from closing for tens of seconds. But subsequent exposure of such an open channel to Mg(2+) ions alone could close it, and the closing rate increased with [Mg(2+)] over the micromolar range (half maximal at approximately 50 microM [Mg(2+)]). A simple interpretation is that channel closing is stoichiometrically coupled to hydrolysis of an ATP molecule that remains tightly associated with the open CFTR channel despite continuous washing. If correct, that ATP molecule appears able to reside for over a minute in the catalytic site that controls channel closing, implying that the site must entrap, or have an intrinsically high apparent affinity for, ATP, even without a Mg(2+) ion. Such stabilization of the open-channel conformation of CFTR by tight binding, or occlusion, of an ATP molecule echoes the stabilization of the active conformation of a G protein by GTP.

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25 Thus, although ATPase activity is diminished 10-20-fold in mutant K464A CFTR, and practically abolished in K1250A CFTR (Ramjeesingh et al., 1999), channel opening rate at millimolar [MgATP] has been reported to be reduced only 2-4-fold in K464A and somewhat more severely (5-10-fold) in K1250A CFTR (Carson et al., 1995; Gunderson and Kopito, 1995; Ramjeesingh et al., 1999); and gating persists even in double mutant K464A/K1250A CFTR channels (Carson et al., 1995). Login to comment
196 Analysis of the temporal asymmetry of changes in character of rapid current blocking events during open bursts of CFTR channels, and their modification by nucleotide analogues and by mutation of NBD2 (K1250A) but not NBD1 (K464A), provided additional evidence that hydrolysis of ATP tightly bound at NBD2 causes the channel to close (Gunderson and Kopito, 1995). Login to comment
246 Nucleotide binding assays (at 0ЊC to prevent hydrolysis) using 8-azidoATP photolabeling show that binding occurs with the same micromolar apparent affinity in wild-type, mutant K1250M, and double mutant K464A/ K1250A, CFTR (Carson et al., 1995). Login to comment
247 These findings, together with the fact that even double mutant K464A/ K1250A CFTR channels open and close at measurable rates (Carson et al., 1995), make it seem unlikely that ATP hydrolysis at either NBD1 or NBD2 is a prerequisite for channel opening. Login to comment

[hide] On the mechanism of MgATP-dependent gating of CFTR... J Gen Physiol. 2003 Jan;121(1):17-36. Vergani P, Nairn AC, Gadsby DC
On the mechanism of MgATP-dependent gating of CFTR Cl- channels.
J Gen Physiol. 2003 Jan;121(1):17-36., [PMID:12508051]

Abstract [show]
CFTR, the product of the gene mutated in cystic fibrosis, is an ATPase that functions as a Cl(-) channel in which bursts of openings separate relatively long interburst closed times (tauib). Channel gating is controlled by phosphorylation and MgATP, but the underlying molecular mechanisms remain controversial. To investigate them, we expressed CFTR channels in Xenopus oocytes and examined, in excised patches, how gating kinetics of phosphorylated channels were affected by changes in [MgATP], by alterations in the chemical structure of the activating nucleotide, and by mutations expected to impair nucleotide hydrolysis and/or diminish nucleotide binding affinity. The rate of opening to a burst (1/tauib) was a saturable function of [MgATP], but apparent affinity was reduced by mutations in either of CFTR's nucleotide binding domains (NBDs): K464A in NBD1, and K1250A or D1370N in NBD2. Burst duration of neither wild-type nor mutant channels was much influenced by [MgATP]. Poorly hydrolyzable nucleotide analogs, MgAMPPNP, MgAMPPCP, and MgATPgammaS, could open CFTR channels, but only to a maximal rate of opening approximately 20-fold lower than attained by MgATP acting on the same channels. NBD2 catalytic site mutations K1250A, D1370N, and E1371S were found to prolong open bursts. Corresponding NBD1 mutations did not affect timing of burst termination in normal, hydrolytic conditions. However, when hydrolysis at NBD2 was impaired, the NBD1 mutation K464A shortened the prolonged open bursts. In light of recent biochemical and structural data, the results suggest that: nucleotide binding to both NBDs precedes channel opening; at saturating nucleotide concentrations the rate of opening to a burst is influenced by the structure of the phosphate chain of the activating nucleotide; normal, rapid exit from bursts occurs after hydrolysis of the nucleotide at NBD2, without requiring a further nucleotide binding step; if hydrolysis at NBD2 is prevented, exit from bursts occurs through a slower pathway, the rate of which is modulated by the structure of the NBD1 catalytic site and its bound nucleotide. Based on these and other results, we propose a mechanism linking hydrolytic and gating cycles via ATP-driven dimerization of CFTR's NBDs.

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4 The rate of opening to a burst (1/␶ib) was a saturable function of [MgATP], but apparent affinity was reduced by mutations in either of CFTR`s nucleotide binding domains (NBDs): K464A in NBD1, and K1250A or D1370N in NBD2. Login to comment
9 However, when hydrolysis at NBD2 was impaired, the NBD1 mutation K464A shortened the prolonged open bursts. Login to comment
32 However, in CFTR the Walker A NBD2 mutation K1250A abolished ATP hydrolysis, whereas the NBD1 mutation K464A simply reduced overall hydrolytic activity (Ramjeesingh et al., 1999); and biochemical studies of Walker B aspartate mutations in CFTR (D572N in NBD1, D1370N in NBD2) have not yet been performed. Login to comment
34 Thus, the K1250A mutation dramatically prolonged burst duration, suggesting that hydrolysis at NBD2 might be coupled to burst termination (Carson et al., 1995; Gunderson and Kopito, 1995), whereas the NBD1 mutations K464A, Q552A, and Q552H somewhat slowed channel opening to a burst, suggesting that NBD1 might be a site of ATP interactions governing opening (Carson et al., 1995; Carson and Welsh 1995). Login to comment
41 We studied in detail the dependence of channel gating on [MgATP], gating in the presence of poorly hydrolyzable nucleotide analogs, as well as the effects of mutating residues in the Walker A (K464A and K1250A) and Walker B motifs (in particular, D1370N in NBD2). Login to comment
52 Amounts of cRNA injected were adjusted to vary the level of expression: up to 40 ng/oocyte was required for high expression of K1250A or K464A/K1250A mutant channels, whereas 0.1-0.25 ng/oocyte sufficed for single channel recordings of WT, K464A, or D1370N channels. Login to comment
89 T A B L E I Kinetic Parameters of WT and Mutant CFTR Channels WT K464A D1370N mean Ϯ SEM n mean Ϯ SEM n mean Ϯ SEM n (A) 5 mM MgATP ϩ 300 nM PKA ␶b 644 Ϯ 63 30 620 Ϯ 58 21 3,768 Ϯ 499 21 ␶ib 1,671 Ϯ 172 30 2,760 Ϯ 439 21 3,588 Ϯ 414 21 1,552 Ϯ 170 19 2,438 Ϯ 483 12 2,849 Ϯ 491 12 ␶F 19.3 Ϯ 2.0 30 20.8 Ϯ 2.1 21 49.9 Ϯ 4.3 21 nF 0.57 Ϯ 0.06 30 0.50 Ϯ 0.06 21 2.76 Ϯ 0.25 21 rCO 0.75 Ϯ 0.06 30 0.50 Ϯ 0.05 21 0.38 Ϯ 0.05 21 0.77 Ϯ 0.08 19 0.54 Ϯ 0.08 12 0.47 Ϯ 0.07 12 rOC 1.95 Ϯ 0.15 30 1.92 Ϯ 0.15 21 0.43 Ϯ 0.07 21 (B) 5 mM MgATP ␶b 338 Ϯ 22 18 309 Ϯ 23 8 1,748 Ϯ 215 17 ␶ib 4,506 Ϯ 497 18 6,752 Ϯ 1314 8 9,503 Ϯ 1440 17 4,454 Ϯ 1382 5 6,928 Ϯ 1666 6 7,584 Ϯ 1967 9 ␶F 23.5 Ϯ 3.2 18 16.1 Ϯ 2.2 8 51.5 Ϯ 6.0 17 nF 0.42 Ϯ 0.05 18 0.39 Ϯ 0.06 8 1.40 Ϯ 0.13 17 rCO 0.27 Ϯ 0.03 18 0.18 Ϯ 0.03 8 0.16 Ϯ 0.03 17 0.33 Ϯ 0.09 5 0.18 Ϯ 0.03 6 0.22 Ϯ 0.05 9 rOC 3.28 Ϯ 0.21 18 3.43 Ϯ 0.25 8 0.75 Ϯ 0.09 17 (C) 50 ␮M MgATP ␶b 355 Ϯ 44 12 323 Ϯ 136 4 1,433 Ϯ 381 4 ␶F 27.3 Ϯ 5.2 12 22.1 Ϯ 4.4 4 46.2 Ϯ 10.8 4 nF 0.38 Ϯ 0.05 12 0.45 Ϯ 0.11 4 1.91 Ϯ 0.34 4 (D) 5 mM MgAMPPNP ␶b 1,619 Ϯ 232 32 271 Ϯ 52 8 ␶F 59.5 Ϯ 6.6 32 26.8 Ϯ 7.7 8 nF 2.40 Ϯ 0.26 32 0.38 Ϯ 0.10 8 Kinetic parameters were obtained using a maximum likelihood simultaneous fit to dwell-time histograms at all conductance levels (Csanády, 2000). Login to comment
113 (B and C) Representative traces for prephosphorylated K464A and D1370N channels. Login to comment
114 Relative opening (D) and closing (E) rates (mean Ϯ SEM, 2 Յ n Յ 7) from analysis of records as in A-C for WT (blue circles), K464A (red triangles), and D1370N (green squares) channels at 10 ␮M Յ [MgATP] Յ 5 mM, plotted on semilogarithmic axes. Login to comment
116 Curves in D show Michaelis-Menten fits, yielding K0.5 of 56 Ϯ 5, 807 Ϯ 185, 391 Ϯ 118 ␮M, and rCOmax of 1.02, 1.16, and 1.08, for WT, K464A, and D1370N, respectively. Login to comment
123 Compared with WT, both K464A (Walker A lysine in NBD1) and D1370N (Walker B aspartate in NBD2) mutant CFTR channels opened less frequently at low [MgATP] (e.g., 50 ␮M; Figs. 2, A-D), and this defect could be largely overcome by raising the [MgATP], so that, at saturating [MgATP], opening rates of WT, K464A, and D1370N channels differed by less than a factor of two (Table I). Login to comment
125 As expected (see below) for channels in which opening rate, but not closing rate, is sensitive to [MgATP], the dependence of Po on [MgATP] was not very different from that of rCO, shown in Fig. 2 D, for WT (see Fig. 3 C), K464A, or D1370N channels. Login to comment
151 Catalytic Site Mutations at NBD1 Do Not Alter Channel Closing from Normal, MgATP-elicited Bursts The average rate of closure of K464A mutant CFTR channels from open bursts was closely similar to that of WT CFTR under comparable conditions (Figs. 4, A-F, and 5, A, B, and E); it was likewise approximately independent of [MgATP] (Fig. 2 E, red triangles) and it was similarly reduced roughly twofold by strong phosphorylation (Fig. 4, D and E; Table I). Login to comment
152 Also like WT, the burst duration distributions of K464A mutant channels were well described by single exponential functions (Fig. 4, D-F). Login to comment
157 Burst duration distributions are similar for WT (A-C) and K464A (D-F) channels under comparable conditions, as indicated (PKA present for left column only). Login to comment
160 Only for K464A at ␮M MgATP (F) could the likelihood be significantly increased by including a second component, though with a shorter (but not longer; Ikuma and Welsh, 2000) mean: ␶1 ϭ 30 ms, a1 ϭ 0.17; ␶2 ϭ 263 ms, a2 ϭ 0.83; increase in log likelihood, ⌬LL ϭ 8.3; number of bursts fitted, M ϭ 263; giving (⌬LL - ln(2M) ϭ 2.0). Login to comment
161 The small differences between means at mM and ␮M MgATP (B vs. C, E vs. F) may be only apparent, as the mean ␶b, estimated by multichannel kinetic fits, from these same stretches of record at ␮M MgATP is not significantly different from that during intervening stretches in 5 mM MgATP (for WT: ␶b␮M/␶b5mM ϭ 1.03 Ϯ 0.07, n ϭ 9; for K464A: ␶b␮M/␶b5mM ϭ 0.95 Ϯ 0.13, n ϭ 7). Login to comment
162 (G and H) Representative traces showing gating of K464A and D1370N channels at 15 ␮M MgATP (after PKA removal). Login to comment
163 Prolonged bursts of K464A channels (Ikuma and Welsh, 2000) are not evident. Login to comment
183 Patches contained one WT (A), K464A (B), or S573E (D) channel, or more than one D572N (C) channel. Login to comment
184 (E) Summary of mean (ϮSEM) ␶b values at 5 mM MgATP and 300 nM PKA (n ϭ 30, 21, 9, and 7 for WT, K464A, D572N, and S573E, respectively). Login to comment
218 Closing from Locked-open Bursts Is Faster for K464A Mutants than for WT Channels Although the K464A mutation did not alter open burst duration of channels exposed to MgATP (Figs. 2 E, 4, and 5), regardless of phosphorylation status (Fig. 4; Table I), it did significantly reduce the duration of certain unusually prolonged bursts. Login to comment
222 For K464A channels, on the other hand (Fig. 10 B), the slow component comprised a somewhat smaller fraction (as ϭ 0.63 Ϯ 0.04, n ϭ 16, Fig. 10 C) of the current Figure 8. Login to comment
227 The smaller fractional amplitude of the slow component for K464A channels can be explained by this observed shortening of their locked-open bursts without the mutation markedly altering the frequency of entry into such bursts. Login to comment
232 The analogous estimate for K464A channels gives an average of 1 locking in every ‫6ف‬ openings. Login to comment
234 The K464A mutation also shortened (Fig. 10, E-G) the similarly prolonged bursts of NBD2 mutant K1250A channels exposed to MgATP alone (Fig. 6 C). Login to comment
242 The K464A mutation speeds exit from locked open burst states. Login to comment
244 (B) Current decay is much faster for the K464A mutant in the same conditions. Login to comment
245 Blue fit lines in A and B show only the slow components of double exponential fits, with ␶s ϭ 67.8s, as ϭ 0.92 for WT, and ␶s ϭ 8.7s, as ϭ 0.79 for K464A. Login to comment
246 (C and D) Summaries of fractional amplitude, as (C), and time constant, ␶s (D), of the slow component from 18 WT and 16 K464A experiments. Login to comment
247 In controls with no MgAMPPNP, closure after exposure to MgATP and PKA yielded ␶ ϭ 1.9 Ϯ 0.2 s (n ϭ 35) for WT and ␶ ϭ 1.0 Ϯ 0.1 s (n ϭ 34) for K464A, and both constructs sometimes showed a small amplitude slower component: for WT, ␶s ϭ 7.6 Ϯ 1.7 s, as ϭ 0.1 Ϯ 0.03 (in 13/35 patches); for K464A, ␶s ϭ 5.9 Ϯ 0.8 s, as ϭ 0.24 Ϯ 0.04 (20/24 patches). Login to comment
249 (F) The additional K464A mutation accelerates channel closure from bursts: for the traces shown, ␶ ϭ 71.7s (K1250A) and ␶ ϭ 29.7s (K464A/K1250A). Login to comment
250 (G) Mean time constants of all 9 K1250A and 9 K464A/K1250A relaxations, each well fit by a single exponential. Login to comment
253 However, in double mutant K464A/K1250A CFTR channels (Fig. 10 F) the current relaxation time constant (␶ ϭ 36 Ϯ 4 s, n ϭ 9), and hence the mean open-burst dwell time, was less than half that of channels bearing the K1250A mutation alone (Fig. 10 G). Login to comment
254 Closing from Bursts During Activation by Poorly Hydrolyzable Analogs Alone Is Faster for K464A Mutants than for WT Channels Like WT CFTR (Fig. 7), mutant K464A channels could be opened by millimolar concentrations of the analogs MgAMPPNP or MgATP␥S alone (Fig. 11), with rates of opening to bursts of 1.5 Ϯ 0.2% (n ϭ 4) at 0.5 mM and 2.9 Ϯ 0.3% (n ϭ 4) at 5 mM MgAMPPNP, and 5.0 Ϯ 0.6% (n ϭ 8) at 2 mM MgATP␥S, of the maximal rate at saturating [MgATP], values not very different from those for WT channels under the same conditions. Login to comment
255 However, the 5-10-fold prolongation of WT bursts by these analogs (Fig. 7 A) was not evident in K464A channels (Fig. 11): for K464A channels opened by MgAMPPNP or MgATP␥S alone, the mean ␶b values were only 1.1 Ϯ 0.2 (n ϭ 8) or 2.2 Ϯ 0.5 (n ϭ 8) times larger, respectively, than at 10 ␮M MgATP. Login to comment
256 Because this consequence of the K464A mutation is manifest during exposure to essentially nonhydrolyzable ATP analogs it cannot be ascribed to any failure of the mutant channel to hydrolyze nucleotide at the NBD1 catalytic site, but instead must be attributed to the alteration of NBD1 structure per se. D I S C U S S I O N We may draw several conclusions from these analyses of gating kinetics of WT and of NBD mutant CFTR channels, in the presence of MgATP and/or of poorly-hydrolyzable analogs: (a) nucleotide binds at both NBD1 and NBD2 catalytic sites before channel opening; (b) the slow opening transition, after nucleotide binding, is highly sensitive to the structures of the beta-␥ phosphate bridging group and of the ␥ phosphate; (c) no further nucleotide binding is required to terminate an open burst; (d) hydrolysis of the nucleotide at NBD2 precedes normal, rapid closing from bursts; (e) if that hydrolysis is prevented, the structure of the NBD1 catalytic site and of the nucleotide bound there can modulate rate of exit from the resulting prolonged (locked) open burst. Login to comment
266 Our results show that mutations within the Walker motifs of either NBD1 (K464A) or NBD2 (D1370N, Figure 11. Login to comment
267 Gating of prephosphorylated K464A channels by poorly hydrolyzable ATP analogs, as indicated. Login to comment
268 Unlike WT (Fig. 7 A), K464A burst duration was not increased during exposure to MgAMPPNP (A and B, ␶b ϭ 270 Ϯ 50 ms, n ϭ 8), and was only slightly increased during exposure to ATP␥S (C, ␶b ϭ 655 Ϯ 170 ms, n ϭ 8), compared with bursts in MgATP (␶b ϭ 276 Ϯ 21 ms, n ϭ 16) in the same patches. Login to comment
269 Note that, due to the lower apparent affinity of K464A for MgATP (Fig. 2), the relative opening rate of mutant channels at 10 ␮M MgATP averaged only 2.3 Ϯ 0.8% (n ϭ 3) of that in saturating MgATP (compared with ‫%11ف‬ for WT), so the opening rate of K464A channels was similar in the presence of millimolar concentrations of the poorly hydrolyzable analogs or of 10 ␮M MgATP. Login to comment
270 K1250A) reduce the apparent affinity of the MgATP binding site(s) involved in channel opening (Figs. 2 and 3), but (at least for K464A and D1370N) affect the maximal opening rate little (Table I). Login to comment
275 Accordingly, although no major difference in [␣32P]8-azidoATP photolabeling at 0ЊC was detected between WT and K464A/K1250A (Carson et al., 1995) or K464A CFTR (Vergani et al., 2002), the K464A mutation alone greatly reduced photolabeling of NBD1 by ␮M [␣32P]8-azidoATP at 37ЊC (Aleksandrov et al., 2002) and virtually abolished stable (i.e., surviving extensive post-incubation washing) photolabeling at 30ЊC (unpublished data). Login to comment
280 Therefore, the simplest interpretation of the reduced apparent affinity with which MgATP elicits opening of K464A and D1370N (and K1250A) mutants compared with WT is that the mutations impair nucleotide binding at two different sites, such that at subsaturating [MgATP] channel opening is limited by MgATP binding at NBD1 in K464A, but at NBD2 in D1370N (and K1250A). Login to comment
286 Allosteric interactions between CFTR`s two NBDs (compare Powe et al., 2002) could, therefore, permit the K464A, D1370N, and K1250A mutations to all affect the same binding site. Login to comment
297 However, we cannot rule out that, at low [MgATP], mutant K464A CFTR channels might open to bursts with only NBD2 occupied by nucleotide. Login to comment
298 In fact, although opening rates for WT and D1370N mutant CFTR channels (Fig. 2 D, blue and green symbols) are satisfactorily described by the Michaelis equation (i.e., opening limited by binding to a single site) the opening rates of K464A channels (Fig. 2 D, red symbols) at low (Յ50 ␮M) [MgATP] are slightly higher than expected. Login to comment
299 If confirmed, these results would be consistent with the right-shifted K464A [MgATP]-rCO curve reflecting principally a reduced nucleotide affinity at NBD1 (now lower than the affinity at NBD2) and a low, but nonzero, opening rate of K464A mutant CFTR channels with nucleotide bound only at the unmodified NBD2 site. Login to comment
300 Therefore, present evidence suggests that nucleotide normally binds to both of WT CFTR`s NBDs before the channel opens, and that opening is limited by nucleotide binding at NBD2 in WT, D1370N, and K1250A CFTR channels, but probably by nucleotide binding at NBD1 in K464A CFTR channels. Login to comment
305 But, by directly comparing gating of the same channels, in the same patch, during exposure to MgAMPPNP, MgAMPPCP, or MgATP␥S, and to MgATP, we find that at concentrations of these analogs expected to be saturating (Figs. 7 and 8; see also Weinreich et al., 1999; Aleksandrov et al., 2001, 2002) the opening rates of WT (Figs. 7-9) and K464A (Fig. 11) channels are only ‫%5ف‬ of that reached at saturating [MgATP]. Login to comment
311 Whereas the K464A mutation in NBD1 has been reported to reduce ‫02ف‬ fold the ATPase activity of purified CFTR (Ramjeesingh et al., 1999), we find that the same mutation diminishes maximal opening rate by only Ͻ50%, similar to effects of other NBD1 catalytic site mutations (Figs. 2 and 5). Login to comment
324 We found no clear dependence of burst duration on [MgATP] (10 ␮M to 5 mM) in WT CFTR (Figs. 2 E, 3 A, and 4, B and C) or in K464A, D1370N, or K1250A mutant channels (Figs. 2 E, 3 B, and 4, E-H), indicating that all ATP binding events precede channel opening and no further binding to the open channel is needed to complete the gating cycle. Login to comment
362 However, the scheme as drawn suggests tight coupling between channel gating and ATP hydrolysis, which is inconsistent with the largely unaltered gating of the catalytically impaired K464A mutant (with ATPase Vmax apparently reduced ‫-02ف‬fold; Ramjeesingh et al., 1999). Login to comment
366 Even higher levels of steady state phosphorylation could prolong normal hydrolytic bursts (Table I, WT and K464A), as well as nonhydrolytic locked-open bursts (Table I, D1370N; Fig. 10 A vs. Fig. 9; Fig. 3 B vs. Fig. 10 E), by stabilizing the open burst states more than the transition states for both possible pathways (forward or backward; Fig. 12 A) for terminating the burst. Login to comment
369 But when hydrolysis (at NBD2) was prevented, by supplying nucleotide resistant to hydrolysis (Figs. 9, and 10, A-D; Fig. 7 vs. Fig. 11), by adding VO4 (Vergani et al., 2002), or by mutating the NBD2 Walker A lysine (K1250A; Fig. 10, E-G), the K464A mutation resulted in less prolonged bursts. Login to comment
370 Very similar reduction of locked-open burst duration by the K464A mutation has been described recently in NIH3T3 and CHO cells (Powe et al., 2002). Login to comment
381 The influence of the K464A mutation seen in Fig. 2 D (also on Po) is then mimicked simply by an ‫-05ف‬fold acceleration of the MgATP dissociation rate from NBD1, together with the Ͻ2-fold observed reduction in maximal opening rate (Table IB). Login to comment
646 Effects on CFTR Cl-channel gating of Walker A lysine mutation K464A imply allosteric interaction between NBDs. Login to comment
639 Effects on CFTR Cl- channel gating of Walker A lysine mutation K464A imply allosteric interaction between NBDs. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochem J. 2003 Apr 15;371(Pt 2):451-62. Annereau JP, Ko YH, Pedersen PL
Cystic fibrosis transmembrane conductance regulator: the NBF1+R (nucleotide-binding fold 1 and regulatory domain) segment acting alone catalyses a Co2+/Mn2+/Mg2+-ATPase activity markedly inhibited by both Cd2+ and the transition-state analogue orthovanadate.
Biochem J. 2003 Apr 15;371(Pt 2):451-62., 2003-04-15 [PMID:12523935]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator), a regulated anion channel and member of the ATP-binding-cassette transporter (ABC transporter) superfamily. Of CFTR's five domains, the first nucleotide-binding fold (NBF1) has been of greatest interest both because it is the major 'hotspot' for mutations that cause CF, and because it is connected to a unique regulatory domain (R). However, attempts have failed to obtain a catalytically active NBF1+R protein in the absence of a fusion partner. Here, we report that such a protein can be obtained following its overexpression in bacteria. The pure NBF1+R protein exhibits significant ATPase activity [catalytic-centre activity (turnover number) 6.7 min(-1)] and an apparent affinity for ATP ( K (m), 8.7 microM) higher than reported previously for CFTR or segments thereof. As predicted, the ATPase activity is inhibited by mutations in the Walker A motif. It is also inhibited by vanadate, a transition-state analogue. Surprisingly, however, the best divalent metal activator is Co(2+), followed by Mn(2+) and Mg(2+). In contrast, Ca(2+) is ineffective and Cd(2+) is a potent inhibitor. These novel studies, while demonstrating clearly that CFTR's NBF1+R segment can act independently as an active, vanadate-sensitive ATPase, also identify its unique cation activators and a new inhibitor, thus providing insight into the nature of its active site.

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44 To obtain ht-NBF1jR mutants in the Walker A region (K464H and K464A), cDNA cassettes were exchanged between BamHI and Bst1107I sites with homologous cDNA cassettes excised from the mutant pMALCR1 MBP-NBF1 plasmids, constructed previously in our laboratory [31]. Login to comment
48 Overexpression of the recombinant ht-NBF1TR (Gly-404-Lys-830) 'wild-type` and mutant proteins (K464H or K464A) Escherichia coli BL21 (DE3) strain, freshly transformed with the 'wild-type` (i.e. recombinant) and mutant ht-NBF1jR plasmids, were grown in 10 ml of LB medium containing 50 µg\ml kanamycin. Login to comment
103 The Walker A motif is underlined, and Lys-464 within this motif is shown in bold to denote the site of mutations (K464H, K464A) made in this study. Login to comment
105 (C) Expression in E. coli of the 'wild-type` ht-NBF1jR protein and mutant forms K464H and K464A. Login to comment
109 Lanes 2, 4 and 6, E. coli cells transformed with plasmids containing, respectively, cDNA encoding 'wild-type` ht-NBF1jR, mutant-form K464H and mutant-form K464A prior to induction with IPTG; lanes 3, 5 and 7, as for lanes 2, 4 and 6 respectively but after induction with 0.3 mM IPTG. Login to comment
110 (D) SDS/PAGE pattern obtained at different stages of purification of the 'wild-type` ht-NBF1jR protein and mutant forms K464H and K464A. Login to comment
113 SDS/PAGE (10%) was used to analyse 10 µl aliquots at each step as follows: lane 1, molecular-mass markers as defined in (C); lanes 2-4, supernatant from guanidine hydrochloride-extracted E. coli overexpressing the 'wild-type` ht-NBF1jR protein and its mutant forms K464A and K464H respectively; lanes 5-7, flow-through fractions of the same extracts after their application to a Ni-NTA column (note that the $ 50 kDa proteins, i.e. the 'wild-type` ht-NBF1jR protein and its mutant forms, were retained); lanes 8-10, eluates derived from Ni-NTA columns containing, respectively, the 'wild-type` ht-NBF1jR protein and its K464A and K464H mutant forms after including 10 mM imidazole in the elution buffer; lanes 11-13, eluates derived from the same Ni-NTA columns after adding 200 mM imidazole to the elution buffer. Login to comment
120 E. coli transformed with the pET-28a(T) expression plasmid encoding NBF1TR residues 404-830, and a short His-tagged N-terminal region, markedly overproduce both 'wild-type` and mutant forms (K464H and K464A) of the protein Results presented in Figure 1(C) summarize SDS\PAGE patterns of E. coli cells harbouring the 'wild-type` and mutant ht-NBF1jR-containing plasmids before and after induction with IPTG. Login to comment
122 The same applies to lane 5 (mutant K464H with IPTG) versus lane 4 (mutant K464H without IPTG), and to lane 7 (mutant K464A with IPTG) versus lane 6 (mutant K464A without IPTG). Login to comment
127 The ht-NBF1TR 'wild-type` protein and its mutant forms can be purified to apparent homogeneity on a Ni-NTA column and show cross-reactivity with a monoclonal antibody to CFTR exhibiting specificity for NBF1 As the overexpressed 'wild-type` and mutant (K464H and K464A) proteins were tagged with six histidines at their N-terminus (see the Materials and methods section and Figure 1B), they were amenable to purification on a column containing bound Ni#+. Login to comment
128 For this reason, the wild-type and mutant E. coli cell pellets derived from the overexpression experiments described above were treated with a buffered solution containing the denaturant guanidine hydrochloride (6 M), and after centri- on the SDS/PAGE gels for the 'wild-type` ht-NBF1jR protein (lane 11) and its mutant forms K464A (lane 12) and K464H (lane 13). Login to comment
129 (E) Cross-reactivity of the purified 'wild-type` ht-NBF1jR protein and mutant form K464A with an anti-CFTR antibody (MATG 1061) with specificity for the NBF1 domain. Login to comment
131 Lanes 1 and 2, purified 'wild-type` ht-NBF1jR protein (0.5 µg) and its K464A mutant (0.5 µg), respectively. Login to comment
142 Data presented in Figure 1(E) show that the purified 'wild-type` ht-NBF1jR protein (lane 1) and its K464A mutant (lane 2) cross-reacted with a monoclonal antibody to CFTR exhibiting specificity for NBF1. Login to comment
152 (B) ATPase activity versus [ATP] plot for the purified renatured 'wild-type` ht-NBF1jR protein and mutant forms K464H and K464A. Login to comment
154 The assay contained, in 75 µl of renaturation buffer, ATP concentrations of 0-100 µM, 5 mM MgCl2 and 0.47 µg of either the 'wild-type` ht-NBF1jR protein (WT) or its mutant forms K464H and K464A, pH 7.5. Login to comment
159 (D) Relative ATPase activities obtained for the 'wild-type` NBF1jR Protein (WT) and mutant forms K464H and K464A using the [32 P]Pi release assay. Login to comment
160 The assay was carried out in 200 µl of renaturation buffer, pH 7.5, containing 3.7 µg of the 'wild-type` ht-NBF1jR protein or its mutant forms (K464H, or K464A), and supplemented with 250 µM [γ-32 P]ATP (1.4i106 c.p.m.) and 1 mM divalent cation. Login to comment
200 Subsequent studies carried out with ht-NBF1jR proteins with mutations in the Walker A consensus region (K464H and K464A) showed that the Vmax was reduced about 50% in each case (Figure 2B). Login to comment

[hide] CFTR directly mediates nucleotide-regulated glutat... EMBO J. 2003 May 1;22(9):1981-9. Kogan I, Ramjeesingh M, Li C, Kidd JF, Wang Y, Leslie EM, Cole SP, Bear CE
CFTR directly mediates nucleotide-regulated glutathione flux.
EMBO J. 2003 May 1;22(9):1981-9., 2003-05-01 [PMID:12727866]

Abstract [show]
Studies have shown that expression of cystic fibrosis transmembrane conductance regulator (CFTR) is associated with enhanced glutathione (GSH) efflux from airway epithelial cells, implicating a role for CFTR in the control of oxidative stress in the airways. To define the mechanism underlying CFTR-associated GSH flux, we studied wild-type and mutant CFTR proteins expressed in Sf9 membranes, as well as purified and reconstituted CFTR. We show that CFTR-expressing membrane vesicles mediate nucleotide-activated GSH flux, which is disrupted in the R347D pore mutant, and in the Walker A K464A and K1250A mutants. Further, we reveal that purified CFTR protein alone directly mediates nucleotide-dependent GSH flux. Interestingly, although ATP supports GSH flux through CFTR, this activity is enhanced in the presence of the non-hydrolyzable ATP analog AMP-PNP. These findings corroborate previous suggestions that CFTR pore properties can vary with the nature of the nucleotide interaction. In conclusion, our data demonstrate that GSH flux is an intrinsic function of CFTR and prompt future examination of the role of this function in airway biology in health and disease.

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2 We show that CFTR-expressing membrane vesicles mediate nucleotide-activated GSH ¯ux, which is disrupted in the R347D pore mutant, and in the Walker A K464A and K1250A mutants. Login to comment
94 To assess the nucleotide dependence of GSH permeation through CFTR, we determined the consequences of lysine mutations in the conserved Walker A consensus motifs for ATP binding in NBD1 and NBD2: K464A and K1250A, respectively. Login to comment
96 In Figure 4, we show that both the K464A and K1250A mutants exhibit similar signi®cant reductions in GSH ¯ux. Login to comment
97 We observed that GSH uptake in both the K464A and K1250A membrane vesicles was 3to 4-fold lower than in vesicles expressing wild-type CFTR protein, yielding permeability values of 132 and 120 pmol/mg CFTR/h, respectively (P < 0.001). Login to comment
102 Membrane vesicles expressing phosphorylated wild-type, K464A or K1250A CFTR were incubated with 20 nM [35S]GSH and 1 mM cold GSH in CFTR transport buffer, in the presence of MgAMPPNP. Login to comment
104 Values shown represent the mean activity (T SEM; for K464A and K1250A, n = 4; for wild-type CFTR, n = 5). Login to comment
105 Inset: expression of CFTR in membranes from Sf9 cells transfected with wild-type, K464A or K1250A CFTR constructs. Login to comment
194 An Sf9 cell pellet (0.5 l) expressing either recombinant CFTR-His proteins (wild type or mutant: R347D, K464A, K1250A) or no CFTR was solubilized in 30 ml of homogenization buffer containing 250 mM sucrose, 50 mM Tris±HCl, 0.25 mM CaCl2 pH 7.5 and protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). Login to comment

[hide] Prolonged nonhydrolytic interaction of nucleotide ... J Gen Physiol. 2003 Sep;122(3):333-48. Basso C, Vergani P, Nairn AC, Gadsby DC
Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating.
J Gen Physiol. 2003 Sep;122(3):333-48., [PMID:12939393]

Abstract [show]
CFTR, the protein defective in cystic fibrosis, functions as a Cl- channel regulated by cAMP-dependent protein kinase (PKA). CFTR is also an ATPase, comprising two nucleotide-binding domains (NBDs) thought to bind and hydrolyze ATP. In hydrolyzable nucleoside triphosphates, PKA-phosphorylated CFTR channels open into bursts, lasting on the order of a second, from closed (interburst) intervals of a second or more. To investigate nucleotide interactions underlying channel gating, we examined photolabeling by [alpha32P]8-N3ATP or [gamma32P]8-N3ATP of intact CFTR channels expressed in HEK293T cells or Xenopus oocytes. We also exploited split CFTR channels to distinguish photolabeling at NBD1 from that at NBD2. To examine simple binding of nucleotide in the absence of hydrolysis and gating reactions, we photolabeled after incubation at 0 degrees C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30 degrees C, with extensive washing, also at 30 degrees C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30 degrees C nucleotide remained tightly bound at NBD1 for many minutes, in the form of nonhydrolyzed nucleoside triphosphate. As nucleotide-dependent gating of CFTR channels occurred on the time scale of seconds under comparable conditions, this suggests that the nucleotide interactions, including hydrolysis, that time CFTR channel opening and closing occur predominantly at NBD2. Vanadate also appeared to act at NBD2, presumably interrupting its hydrolytic cycle, and markedly delayed termination of channel open bursts. Vanadate somewhat increased the magnitude, but did not alter the rate, of the slow loss of nucleotide tightly bound at NBD1. Kinetic analysis of channel gating in Mg8-N3ATP or MgATP reveals that the rate-limiting step for CFTR channel opening at saturating [nucleotide] follows nucleotide binding to both NBDs. We propose that ATP remains tightly bound or occluded at CFTR's NBD1 for long periods, that binding of ATP at NBD2 leads to channel opening wherupon its hydrolysis prompts channel closing, and that phosphorylation acts like an automobile clutch that engages the NBD events to drive gating of the transmembrane ion pore.

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46 We have previously described pGEMHE-CFTR, pGEMHE-Flag-3-835 (Chan et al., 2000), and pGEMHE-K464A (Vergani et al., 2003). Login to comment
82 Oocytes were injected with 0.1-10 ng of cRNA transcribed from pGEMHE-CFTR (WT or K464A). Login to comment
231 The K464A Mutation Little Influences Binding at 0ЊC, but Diminishes Occlusion at 30ЊC, at Low [Nucleotide] Because the mutation K464A at the Walker A lysine in NBD1 impairs CFTR maturation in mammalian cells (e.g., Aleksandrov et al., 2001), we compared nucleotide interactions with WT and K464A CFTR expressed in oocytes, in which we also compared electrophysiological responses. Login to comment
232 Despite loading twice as much membrane protein in the K464A lanes as in the WT lanes (Fig. 8 A, right) to compensate for diminished K464A expression (Fig. 8 A, left), negligible occlusion of [␣32P]8-N3ATP at 30ЊC was observed for K464A CFTR, either with or without Vi. Login to comment
233 In contrast, simple binding of 5 or 50 ␮M [␣32P]8-N3ATP, assayed by photolabeling in oocyte membranes at 0ЊC, seemed little different for Flag-K464A than for Flag-WT CFTR (Fig. 8 B), even though that binding occurs predominantly at NBD1 (Fig. 3, above), the structurally altered catalytic site. Login to comment
234 The fact that opening of K464A CFTR channels is impaired at low ␮M [MgATP] (Vergani et al., 2003) suggests that channel opening and nucleotide occlusion at NBD1 share a common step that occurs after the simple association with nucleotide. Login to comment
235 The K464A Mutation Virtually Eliminates the Vi-dependent Slowing of CFTR Channel Closing Unlike the slow decline of roughly half of the WT CFTR current (Fig. 4, above), the macroscopic current flowing through K464A channels activated by PKA, MgATP, and Vi decayed relatively rapidly upon nucleotide withdrawal (Fig. 8 C). Login to comment
236 The current relaxation after exposure to Vi had a double exponential time course (smooth fit line, Fig. 8 C, center): most of the current decayed rapidly (␶fast ϭ 0.9 Ϯ 0.1 s, n ϭ 10) with a time constant like that of closing (␶b ϭ 620 Ϯ 58 ms, n ϭ 24; Vergani et al., 2003) of K464A channels exposed to just PKA and 5 mM MgATP, indicating that the majority of K464A CFTR channels did not respond to the presence of Vi. Login to comment
250 K464A CFTR, mutated at the Walker A lysine in NBD1, binds but does not occlude micromolar concentrations of 8-N3ATP, in accord with our conclusion from analyses of channel gating that the mutation lowers the apparent affinity for nucleotide interactions required for the transition to the channel open-burst state. Login to comment
257 Mutation K464A in CFTR impairs occlusion at 30ЊC, but not binding at 0ЊC, at low [nucleotide], and disrupts Vi-induced stabilization of open burst state. Login to comment
258 (A, left) Membranes of oocytes expressing WT or K464A CFTR (without Flag tags) were run on SDS-PAGE gels, transferred to nitrocellulose membranes, and blotted with anti-R-domain antibody. Login to comment
259 WT CFTR was expressed at least twice as well as K464A CFTR in this batch of oocytes (arrow marks mature fully-glycosylated CFTR; lower, sharper band is core-glycosylated CFTR). Login to comment
261 Twice as much membrane was used for K464A samples as for WT samples. Login to comment
262 (B, left) Immunoblots of membranes from Flag-WTand Flag-K464A-expressing oocytes (the Flag tags facilitated immunoprecipitation to enhanced signal-to-noise ratio, as membranes were not washed before photocrosslinking) blotted with anti-R-domain antibody as in A. Login to comment
263 The membranes contained about one third more WT than mutant K464A protein. Login to comment
265 Approximately 30% more membrane was used for Flag-K464A samples as for Flag-WT samples. Login to comment
266 (C) Macroscopic current in an oocyte patch containing hundreds of K464A CFTR channels. Login to comment
270 (D and E) Mean fit parameters for the time constant (D) and fractional amplitude (E) of the slow component of current decay after activation by 5 mM MgATP plus 300 nM PKA without (white bars) or with 5 mM Vi (black bars) for WT (n ϭ 19) or K464A (n ϭ 10). Login to comment
299 Second, purified NBD1 mutant, K464A, CFTR was reported to hydrolyze MgATP at a maximal rate 10-20-fold lower than that of wild-type CFTR, whereas the equivalent mutation in NBD2, K1250A, essentially abolished hydrolysis (Ramjeesingh et al., 1999). Login to comment
300 Unless the specific activity of that K464A CFTR was greatly underestimated (due to unrecognized nonfunctional K464A protein), if NBD1 is essentially catalytically inactive in intact CFTR as we propose, a possible explanation for the reduced hydrolysis by K464A CFTR is that this mutation somehow impairs MgATP hydrolysis at the NBD2 catalytic site. Login to comment
301 Gating measurements offer no support for this possibility, as the K464A mutation little affects normal CFTR-channel gating (Carson et al., 1995; Gunderson and Kopito, 1995; Powe et al., 2002; Vergani et al., 2003) other than reducing the apparent affinity for MgATP (Vergani et al., 2003). Login to comment
303 Although this destabilization could reflect loss of an allosteric influence of the wild-type NBD1 catalytic site on the NBD2 catalytic site, if transition to the open burst state involves formation of an NBD1/NBD2 dimer with interfacial catalytic sites (Vergani et al., 2003), then the K464A mutation could act locally within the interface to reduce stability of the NBD dimer. Login to comment

[hide] Structure of nucleotide-binding domain 1 of the cy... EMBO J. 2004 Jan 28;23(2):282-93. Epub 2003 Dec 18. Lewis HA, Buchanan SG, Burley SK, Conners K, Dickey M, Dorwart M, Fowler R, Gao X, Guggino WB, Hendrickson WA, Hunt JF, Kearins MC, Lorimer D, Maloney PC, Post KW, Rajashankar KR, Rutter ME, Sauder JM, Shriver S, Thibodeau PH, Thomas PJ, Zhang M, Zhao X, Emtage S
Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator.
EMBO J. 2004 Jan 28;23(2):282-93. Epub 2003 Dec 18., 2004-01-28 [PMID:14685259]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.

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42 A mutated version, K464A, which was expected to have reduced ATP binding, was also cloned and purified in the same manner and was used for ATP-binding measurements (see below). Login to comment
149 ATP binding was significantly reduced by the K464A mutation, presumably because electrostatic interactions with the band g-phosphates of ATP (Figure 4A) are lost. Login to comment
201 Wild type (K), K464A mutant (J), and ATP binding to the filters in the absence of protein (.) Login to comment

[hide] Nucleotide-binding domains of human cystic fibrosi... Cell Mol Life Sci. 2004 Jan;61(2):230-42. Callebaut I, Eudes R, Mornon JP, Lehn P
Nucleotide-binding domains of human cystic fibrosis transmembrane conductance regulator: detailed sequence analysis and three-dimensional modeling of the heterodimer.
Cell Mol Life Sci. 2004 Jan;61(2):230-42., [PMID:14745501]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is encoded by the gene that is defective in cystic fibrosis, the most common lethal inherited disease among the Caucasian population. CFTR belongs to the ABC transporter superfamily, whose members form macromolecular architectures composed of two membrane-spanning domains and two nucleotide-binding domains (NBDs). The experimental structures of NBDs from several ABC transporters have recently been solved, opening new avenues for understanding the structure/function relationships and the consequences of some disease-causing mutations of CFTR. Based on a detailed sequence/structure analysis, we propose here a three-dimensional model of the human CFTR NBD heterodimer. This model, which is in agreement with recent experimental data, highlights the specific features of the CFTR asymmetric active sites located at the interface between the two NBDs. Moreover, additional CFTR-specific features can be identified at the subunit interface, which may play critical roles in active site interdependence and are uncommon in other NBD dimers.

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235 These results are in agreement with the recent work of Aleksandrov et al. [54] with the Walker A lysine mutants K464A and K1250A, with the finding that NBD2 is a site of rapid nucleotide turnover, while NBD1 is a site of stable nucleotide interaction. Login to comment

[hide] A heteromeric complex of the two nucleotide bindin... J Biol Chem. 2004 Oct 1;279(40):41664-9. Epub 2004 Jul 28. Kidd JF, Ramjeesingh M, Stratford F, Huan LJ, Bear CE
A heteromeric complex of the two nucleotide binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) mediates ATPase activity.
J Biol Chem. 2004 Oct 1;279(40):41664-9. Epub 2004 Jul 28., 2004-10-01 [PMID:15284228]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a member of the ABC superfamily of transporter proteins. Recently, crystal structures of intact, prokaryotic members of this family have been described. These structures suggested that ATP binding and hydrolysis occurs at two sites formed at the interface between their nucleotide binding domains (NBDs). In contrast to the prokaryotic family members, the NBDs of CFTR are asymmetric (both structurally and functionally), and previous to the present studies, it was not clear whether both NBDs are required for ATP hydrolysis. In order to assess the relative roles of the two NBDs of human CFTR, we purified and reconstituted NBD1 and NBD2, separately and together. We found that NBD1 and NBD2 by themselves exhibited relatively low ATPase activity. Co-assembly of NBD1 and NBD2 exhibited a 2-3-fold enhancement in catalytic activity relative to the isolated domains and this increase reflected enhanced ATP turnover (V(max)). These data provide the first direct evidence that heterodimerization of the NBD1 and NBD2 domains of CFTR is required to generate optimal catalytic activity.

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188 Mutation of the canonical Walker A lysine in either NBD1 (K464A, targeting Site A) or NBD2 (K1250A, targeting Site B) decreased ATPase activity of the whole protein by greater than 50% (39). Login to comment
193 Our data suggest that the nonconventional site to which Lys464 contributes (Site A) also participates in the overall activity, since the K464A mutation causes a significant, albeit partial, decrease in function of the full-length protein. Login to comment

[hide] Normal gating of CFTR requires ATP binding to both... Proc Natl Acad Sci U S A. 2005 Jan 11;102(2):455-60. Epub 2004 Dec 27. Berger AL, Ikuma M, Welsh MJ
Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain.
Proc Natl Acad Sci U S A. 2005 Jan 11;102(2):455-60. Epub 2004 Dec 27., 2005-01-11 [PMID:15623556]

Abstract [show]
ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [alpha-(32)P]8-N(3)-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating.

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6 We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. Login to comment
71 The 6% gels revealed that CFTR-A462F and -K464A produced little band C protein, whereas other variants produced predominantly the band C form. Login to comment
123 In CFTR, it has been reported that the K1250A and K464A mutations prevented [␣-32 P]8-N3-ATP photolabeling of the respective NBD (14, 15), whereas another study found that K464A did not prevent [␣-32 P]8-N3-ATP NBD1 photolabeling (36). Login to comment
124 We found that neither the K1250A nor K464A mutations prevented [␣-32 P]8-N3-ATP photolabeling of the NBDs (Fig. 3A). Login to comment
132 (A) Autoradiogram of [␣-32P]8-N3-ATP labeling of CFTR-K464A and K1250A; labeling of both NBDs was observed for each mutant. Login to comment
164 We found that the K464A mutation did not prevent [␣-32 P]8-N3-ATP photolabeling of NBD1 or NBD2 (Fig. 3A), a finding consistent with some (36) but not all earlier work (14, 15). Login to comment
165 Our finding indicates that ATP can bind NBD1 bearing the K464A mutation, although for the reasons described above, the ATP binding affinity might have been reduced. Login to comment
166 We reasoned that if ATP binds CFTR-K464A to increase opening, then NEM modification of A462C͞K464A should block binding and reduce activity. Login to comment
167 On the other hand, if the K464A mutation prevents ATP binding (contrary to our labeling results), then NEM modification should have no additional effect on current. Login to comment
169 Therefore, we conclude that ATP binding to NBD1 played an important role in channel activity even when it contained the K464A mutation. Login to comment
172 The S1248F mutation will block NBD2 ATP binding, thereby limiting ATP interactions to NBD1, and the K464A mutation will prevent NBD1 ATP hydrolysis. Login to comment
177 Thus, when ATP binding to NBD2 was eliminated, we found that the K464A mutation had little effect on gating. Login to comment
205 Third, earlier observations showed that the K464A mutation, which blocks hydrolysis, had relatively minor effects on CFTR gating (16-19, 21). Login to comment
207 For example, our results and those of Basso et al. (36) showed that [␣-32 P]8-N3-ATP labeled the K464A NBD1, although perhaps with a reduced affinity, whereas Aleksandrov et al. (14, 15) reported that it ablated labeling. Login to comment

[hide] CFTR gating II: Effects of nucleotide binding on t... J Gen Physiol. 2005 Apr;125(4):377-94. Epub 2005 Mar 14. Bompadre SG, Cho JH, Wang X, Zou X, Sohma Y, Li M, Hwang TC
CFTR gating II: Effects of nucleotide binding on the stability of open states.
J Gen Physiol. 2005 Apr;125(4):377-94. Epub 2005 Mar 14., [PMID:15767296]

Abstract [show]
Previously, we demonstrated that ADP inhibits cystic fibrosis transmembrane conductance regulator (CFTR) opening by competing with ATP for a binding site presumably in the COOH-terminal nucleotide binding domain (NBD2). We also found that the open time of the channel is shortened in the presence of ADP. To further study this effect of ADP on the open state, we have used two CFTR mutants (D1370N and E1371S); both have longer open times because of impaired ATP hydrolysis at NBD2. Single-channel kinetic analysis of DeltaR/D1370N-CFTR shows unequivocally that the open time of this mutant channel is decreased by ADP. DeltaR/E1371S-CFTR channels can be locked open by millimolar ATP with a time constant of approximately 100 s, estimated from current relaxation upon nucleotide removal. ADP induces a shorter locked-open state, suggesting that binding of ADP at a second site decreases the locked-open time. To test the functional consequence of the occupancy of this second nucleotide binding site, we changed the [ATP] and performed similar relaxation analysis for E1371S-CFTR channels. Two locked-open time constants can be discerned and the relative distribution of each component is altered by changing [ATP] so that increasing [ATP] shifts the relative distribution to the longer locked-open state. Single-channel kinetic analysis for DeltaR/E1371S-CFTR confirms an [ATP]-dependent shift of the distribution of two locked-open time constants. These results support the idea that occupancy of a second ATP binding site stabilizes the locked-open state. This binding site likely resides in the NH2-terminal nucleotide binding domain (NBD1) because introducing the K464A mutation, which decreases ATP binding affinity at NBD1, into E1371S-CFTR shortens the relaxation time constant. These results suggest that the binding energy of nucleotide at NBD1 contributes to the overall energetics of the open channel conformation.

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11 This binding site likely resides in the NH2-terminal nucleotide binding domain (NBD1) because introducing the K464A mutation, which decreases ATP binding affinity at NBD1, into E1371S-CFTR shortens the relaxation time constant. Login to comment
35 Previous studies suggested that ATP hydrolysis at NBD1 was involved in the opening of the channel since mutations of the Walker A lysine at NBD1 (e.g., K464A) decrease the channel opening rate (Carson et al., 1995; Gunderson and Kopito, 1995; cf. Login to comment
37 However, Powe et al. (2002) reexamined the gating kinetics of K464A mutant CFTR and found little difference in the opening rate between this mutant and WT-CFTR. Login to comment
58 Similarly, the 0.9-kb PflMI-EcoRV fragments were used to replace the corresponding ones in pcDNA 3.1 WT-CFTR or pcDNA 3.1 K464A-CFTR (Powe et al., 2002) to generate pcDNA3.1 D1370N, pcDNA 3.1 E1371S, and pcDNA3.1 K464A/ E1371S CFTR constructs. Login to comment
247 Current Relaxations of the K464A/E1371S Mutant According to the most recent model (Scheme 2 in the accompanying paper) for CFTR gating (Vergani et al., 2003), NBD1 has little role in the gating transitions since the off rate is extremely slow, and ATP binding at NDB2 precedes channel opening. Login to comment
262 The K464A mutation shortens the locked-open time of E1371S-CFTR. Login to comment
263 (A) Sample trace of K464A/E1371S-CFTR channels in the presence of 1 mM ATP ϩ PKA. Login to comment
267 (C) Sample trace of K464A/E1371S-CFTR channels in the presence of 10 ␮M ATP (blue curve). Login to comment
273 Since the K464 mutation has a mild trafficking defect (Cheng et al., 1990; unpublished data), and ⌬R-CFTR already suffers from low expression, we decided to make the K464A/E1371S double mutant construct in the WT background. Login to comment
275 The current decay of the K464A/E1371S-CFTR channel currents is indeed faster than that of E1371S-CFTR, resulting in a shorter relaxation time constant (19.60 Ϯ 0.01 s) upon washout of 1 mM ATP (Fig. 10 B). Login to comment
276 This relaxation time constant is even shorter when the K464A/E1371S-CFTR channel is opened with 10 ␮M ATP (13.95 Ϯ 0.02 s). Login to comment
277 Since the number of K464A/E1371S-CFTR channels is relatively low due to a moderate trafficking defect, it is easier to observe microscopic channel behavior at 10 ␮M ATP (Fig. 10 C). Login to comment
278 As shown previously for ⌬R/E1371S-CFTR (Fig. 5), the current trace reveals that K464A/E1371S-CFTR channels also exhibit numerous brief openings that last for tens to hundreds of milliseconds in the presence of 10 ␮M ATP. Login to comment
366 Powe et al. (2002) showed that the K464A mutation shortens the open time by 40% at high [ATP]. Login to comment
367 Interestingly, introducing the K464A mutations into the K1250A construct significantly decreases the locked-open time (Powe et al., 2002; Vergani et al., 2003). Login to comment
368 An equivalent observation is also made in the current report for K464A/E1371S mutants. Login to comment
371 As mentioned above, Powe et al. (2002) showed that the K464A mutant exhibits a normal opening rate despite a lower ATP binding affinity at NBD1 for this mutant. Login to comment
380 This hypothesis is based on the observation that mutations that affect ATP binding at NBD1 (e.g., K464A) alter the stability of the open state of K1250A, suggesting an interaction between two ATP-binding sites. Login to comment
405 Although we proposed that ATP binding at NBD2 plays a critical role in channel opening (see above), this idea is based more on default since we observed that the K464A mutation, which decreases ATP binding affinity at NBD1 (Basso et al., 2003), does not affect the opening rate (Powe et al., 2002). Login to comment

[hide] High affinity ATP/ADP analogues as new tools for s... J Physiol. 2005 Dec 1;569(Pt 2):447-57. Epub 2005 Oct 13. Zhou Z, Wang X, Li M, Sohma Y, Zou X, Hwang TC
High affinity ATP/ADP analogues as new tools for studying CFTR gating.
J Physiol. 2005 Dec 1;569(Pt 2):447-57. Epub 2005 Oct 13., 2005-12-01 [PMID:16223764]

Abstract [show]
Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N6-modified ATP analogues tested, N6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K(1/2) of 1.6 +/- 0.4 microm (>50-fold more potent than ATP). The maximal open probability (P(o)) in the presence of P-ATP was approximately 30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating.

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220 Our earlier studies show that a mutation (i.e. K464A) that causes a decrease of ATP binding affinity at the NBD1 site (Basso et al. 2003) does not affect the opening rate or the ATP dose-response relationship (Powe et al. 2002; cf. Vergani et al. 2003), suggesting ATP binding at the NBD1 site may not be absolutely required for channel opening (also see Bompadre et al. 2005b). Login to comment
222 The proposition that P-ATP acts on the NBD1 site to modulate channel closing is also based on our earlier studies with the K464A mutant. Login to comment
223 Although this mutation does not affect channel opening, K464A-CFTR exhibits a shorter open time (Powe et al. 2002). Login to comment
224 In addition, K464A mutation decreases the locked open time of hydrolysis-deficient mutants K464A/K1250A and K464A/E1371S (Powe et al. 2002; Vergani et al. 2003; Bompadre et al. 2005b), supporting the idea that the strength of ligand binding at the NBD1 site affects the stability of the open state. Login to comment

[hide] State-dependent chemical reactivity of an engineer... J Biol Chem. 2005 Dec 23;280(51):41997-2003. Epub 2005 Oct 14. Zhang ZR, Song B, McCarty NA
State-dependent chemical reactivity of an engineered cysteine reveals conformational changes in the outer vestibule of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2005 Dec 23;280(51):41997-2003. Epub 2005 Oct 14., 2005-12-23 [PMID:16227620]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are gated by binding and hydrolysis of ATP at the nucleotide-binding domains (NBDs). We used covalent modification of CFTR channels bearing a cysteine engineered at position 334 to investigate changes in pore conformation that might accompany channel gating. In single R334C-CFTR channels studied in excised patches, modification by [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET+), which increases conductance, occurred only during channel closed states. This suggests that the rate of reaction of the cysteine was greater in closed channels than in open channels. R334C-CFTR channels in outside-out macropatches activated by ATP alone were modified with first order kinetics upon rapid exposure to MTSET+. Modification was much slower when channels were locked open by the addition of nonhydrolyzable nucleotide or when the R334C mutation was coupled to a second mutation, K1250A, which greatly decreases channel closing rate. In contrast, modification was faster in R334C/K464A-CFTR channels, which exhibit prolonged interburst closed states. These data indicate that the reactivity of the engineered cysteine in R334C-CFTR is state-dependent, providing evidence of changes in pore conformation coupled to ATP binding and hydrolysis at the NBDs. The data also show that maneuvers that lock open R334C-CFTR do so by locking channels into the prominent s2 subconductance state, suggesting that the most stable conducting state of the pore reflects the fully occupied, prehydrolytic state of the NBDs.

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6 In contrast, modification was faster in R334C/K464A-CFTR channels, which exhibit prolonged interburst closed states. Login to comment
45 R334C-, R334C/ K464A-, and R334C/K1250A-CFTR channels were activated by excision into intracellular solution containing 300 mM NMDG-Cl, 1.1 mM MgCl2, 2 mM Tris-EGTA, 1 mM MgATP, 10 mM TES (pH 7.4), 50 units/ml PKA. Login to comment
145 Mutation K464A in NBD1 leads to a great reduction in the channel opening rate. Login to comment
146 We studied outside-out macropatches from oocytes expressing R334C/K1250A- or R334C/K464A-CFTR to determine the effects of these gating domain mutations on the kinetics of modification, using experimental procedures similar to those described above. Login to comment
165 MTSET؉ -induced modification of R334C/K1250A-CFTR and R334C/ K464A-CFTR. Login to comment
166 Shown are outside-out macropatches from oocytes expressing either R334C/K1250A-CFTR (A and C) or R334C/K464A-CFTR (B). Login to comment
171 In contrast, the kinetics of modification of R334C/K464A-CFTR by MTSETϩ were fit best with a first-order exponential function, with ␶ ϭ 1.92 s in this experiment. Login to comment
191 Therefore, we recorded from giant outside-out patches pulled from oocytes expressing R334C/ K464A-CFTR, which would reduce Po considerably by prolonging the interburst closed durations (21-23) (Fig. 5B). Login to comment
192 The macroscopic current of R334C/K464A-CFTR was increased rapidly upon application of 10 ␮M MTSETϩ . Login to comment
194 The modification rate coefficient for MTSETϩ in R334C/K464A-CFTR was 41,864 Ϯ 4,229 M -1 s-1 , which is roughly 2-fold higher than that in R334C-CFTR under identical conditions (p ϭ 0.007). Login to comment
234 Under conditions that decrease channel activity (R334C/K464A-CFTR), the rate of modification was increased dramatically. Login to comment

[hide] Control of the CFTR channel's gates. Biochem Soc Trans. 2005 Nov;33(Pt 5):1003-7. Vergani P, Basso C, Mense M, Nairn AC, Gadsby DC
Control of the CFTR channel's gates.
Biochem Soc Trans. 2005 Nov;33(Pt 5):1003-7., [PMID:16246032]

Abstract [show]
Unique among ABC (ATP-binding cassette) protein family members, CFTR (cystic fibrosis transmembrane conductance regulator), also termed ABCC7, encoded by the gene mutated in cystic fibrosis patients, functions as an ion channel. Opening and closing of its anion-selective pore are linked to ATP binding and hydrolysis at CFTR's two NBDs (nucleotide-binding domains), NBD1 and NBD2. Isolated NBDs of prokaryotic ABC proteins form homodimers upon binding ATP, but separate after hydrolysis of the ATP. By combining mutagenesis with single-channel recording and nucleotide photolabelling on intact CFTR molecules, we relate opening and closing of the channel gates to ATP-mediated events in the NBDs. In particular, we demonstrate that two CFTR residues, predicted to lie on opposite sides of its anticipated NBD1-NBD2 heterodimer interface, are energetically coupled when the channels open but are independent of each other in closed channels. This directly links ATP-driven tight dimerization of CFTR's cytoplasmic NBDs to opening of the ion channel in the transmembrane domains. Evolutionary conservation of the energetically coupled residues in a manner that preserves their ability to form a hydrogen bond argues that this molecular mechanism, involving dynamic restructuring of the NBD dimer interface, is shared by all members of the ABC protein superfamily.

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39 To determine which of the two composite sites in the NBD1-NBD2 dimer is involved in channel opening, we introduced mutations at residues seen to interact directly with the bound nucleotide in the solved crystal structures, in the head of either NBD1 [K464A (Lys464 → Ala)] or NBD2 (D1370N). Login to comment

[hide] Nucleotide-binding domains of cystic fibrosis tran... J Biol Chem. 2006 Feb 17;281(7):4058-68. Epub 2005 Dec 16. Gross CH, Abdul-Manan N, Fulghum J, Lippke J, Liu X, Prabhakar P, Brennan D, Willis MS, Faerman C, Connelly P, Raybuck S, Moore J
Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis.
J Biol Chem. 2006 Feb 17;281(7):4058-68. Epub 2005 Dec 16., 2006-02-17 [PMID:16361259]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl(-) ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [(33)P]phosphate is observed from the gamma-(33)P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to both human NBDs, as the Walker A box lysine mutations abolish this activity. At low protein concentration, the NBDs display an initial slower nonlinear phase in AK activity, suggesting that the activity results from homodimerization. Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. hNBD1 and hNBD2 mixing experiments resulted in an 8-57-fold synergistic enhancement in AK activity suggesting heterodimer formation, which supports a common theme in ABC transporter models. A CFTR gating mechanism model based on adenylate kinase activity is proposed.

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240 Next, we wanted to examine three key CFTR residues in mNBD1 (K464A, G551D, and ⌬F508) to determine what effect these mutations had on their ability to form homodimers or alter AK activity. Login to comment
241 All three mutant proteins overexpressed, however, during their purifications two mutants (⌬F508 and K464A) were problematic. Login to comment
244 Because of the chaperone contamination we were forced to abandon our analysis of the K464A and ⌬F508 proteins. Login to comment

[hide] The ABC protein turned chloride channel whose fail... Nature. 2006 Mar 23;440(7083):477-83. Gadsby DC, Vergani P, Csanady L
The ABC protein turned chloride channel whose failure causes cystic fibrosis.
Nature. 2006 Mar 23;440(7083):477-83., 2006-03-23 [PMID:16554808]

Abstract [show]
CFTR chloride channels are encoded by the gene mutated in patients with cystic fibrosis. These channels belong to the superfamily of ABC transporter ATPases. ATP-driven conformational changes, which in other ABC proteins fuel uphill substrate transport across cellular membranes, in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.

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139 But they are 11 22 11 2 a b ATP ATP ATP ATP C2 Open Concentration of MgATP in µM Relativeopeningrate K1250A K464A C1C0 101 1.0 WT K464A K1250A 0.5 0 102 103 104 Figure 3 | The conserved Walker A lysine is critical for ATP binding in each NBD. Login to comment
144 ATP first binds to the non-mutant site, that is to the NBD2 site (blue) in K464A channels (upper row), but to the NDB1 site (green) in K1250A channels (lower row). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Sheng Li Xue Bao. 2007 Aug 25;59(4):431-42. Bompadre SG, Hwang TC
Cystic fibrosis transmembrane conductance regulator: a chloride channel gated by ATP binding and hydrolysis.
Sheng Li Xue Bao. 2007 Aug 25;59(4):431-42., 2007-08-25 [PMID:17700963]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP-binding cassette (ABC) transporter superfamily. Defective function of CFTR is responsible for cystic fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasian populations. The disease is manifested in defective chloride transport across the epithelial cells in various tissues. To date, more than 1400 different mutations have been identified as CF-associated. CFTR is regulated by phosphorylation in its regulatory (R) domain, and gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBD1 and NBD2). Recent studies reveal that the NBDs of CFTR may dimerize as observed in other ABC proteins. Upon dimerization of CFTR's two NBDs, in a head-to-tail configuration, the two ATP-binding pockets (ABP1 and ABP2) are formed by the canonical Walker A and B motifs from one NBD and the signature sequence from the partner NBD. Mutations of the amino acids that interact with ATP reveal that the two ABPs play distinct roles in controlling ATP-dependent gating of CFTR. It was proposed that binding of ATP to the ABP2, which is formed by the Walker A and B in NBD2 and the signature sequence in NBD1, is critical for catalyzing channel opening. While binding of ATP to the ABP1 alone may not increase the opening rate, it does contribute to the stabilization of the open channel conformation. Several disease-associated mutations of the CFTR channel are characterized by gating defects. Understanding how CFTR's two NBDs work together to gate the channel could provide considerable mechanistic information for future pharmacological studies, which could pave the way for tailored drug design for therapeutical interventions in CF.

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133 [51] showed that, in contrast to previous studies[25,27] , the K464A mutation does not affect the channel opening rate, but it shortens the mean open time. Login to comment
135 In addition, introducing the K464A mutation into the K1250A mutant whose ATP hydrolysis at NBD2 is diminished[25,27] dramatically shortens the stable open state seen in the K1250A mutation (Fig.3). Login to comment
160 This conclusion was reached after finding that the ATP dose-response relationships of the Walker A mutants K464A and K1250A and the Walker B mutant D1370N were shifted towards higher [ATP] com- paredto theATPdose-response curvefor wild-typechannels. Login to comment
164 The K464A mutation shortens current relaxation of K1250A-CFTR. Login to comment
165 A: Representative traces for the current relaxation of K1250A-CFTR and K464A/K1250A-CFTR upon withdrawal of ATP and PKA. Login to comment
183 This same effect was observed previously for K464A by Powe et al[51] . Login to comment

[hide] CLC-0 and CFTR: chloride channels evolved from tra... Physiol Rev. 2008 Apr;88(2):351-87. Chen TY, Hwang TC
CLC-0 and CFTR: chloride channels evolved from transporters.
Physiol Rev. 2008 Apr;88(2):351-87., [PMID:18391167]

Abstract [show]
CLC-0 and cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels play important roles in Cl(-) transport across cell membranes. These two proteins belong to, respectively, the CLC and ABC transport protein families whose members encompass both ion channels and transporters. Defective function of members in these two protein families causes various hereditary human diseases. Ion channels and transporters were traditionally viewed as distinct entities in membrane transport physiology, but recent discoveries have blurred the line between these two classes of membrane transport proteins. CLC-0 and CFTR can be considered operationally as ligand-gated channels, though binding of the activating ligands appears to be coupled to an irreversible gating cycle driven by an input of free energy. High-resolution crystallographic structures of bacterial CLC proteins and ABC transporters have led us to a better understanding of the gating properties for CLC and CFTR Cl(-) channels. Furthermore, the joined force between structural and functional studies of these two protein families has offered a unique opportunity to peek into the evolutionary link between ion channels and transporters. A promising byproduct of this exercise is a deeper mechanistic insight into how different transport proteins work at a fundamental level.

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732 They showed that purified K464A-CFTR exhibits a nearly identical opening rate as wild-type channels at 1 mM ATP. Login to comment
734 (237) not only confirmed this result, but also showed that the relationship between [ATP] and the opening rate is not affected by the K464A mutation, thus casting serious doubt on the role of ATP binding at ABP1 in channel opening. Login to comment
794 Although the mutations that presumably decrease ATP affinity at the ABP1 (K464A and W401G) have questionable effects on the ability of ATP to increase the opening rate of CFTR, both mutants show a shortened open time, suggesting a destabilization of the open state by the mutations (237, 360). Login to comment
795 The effect of K464A on the open time was seen even in early studies (42, 251). Login to comment
796 The shortened opening bursts of K464A-CFTR, compared with wild-type channels, are visually discernable in Ramjeesingh et al. Login to comment
800 (324) reported that the open time for K464A-CFTR is not significantly different from that of wild-type channels. Login to comment
802 It is, however, important to note that the K464A mutation does shorten the locked open time of hydrolysis-deficient mutants in two different studies (237, 324), suggesting that this effect on the open time is not due to a potential allosteric action of the mutations at the ABP1 on the ATP hydrolysis rate at the ABP2, which normally determines the rate of channel closing. Login to comment
804 Thus a reduction of the free energy of ATP binding could be reported as a decreased open-time constant as seen with the K464A and W401G mutants. Login to comment

[hide] Review. ATP hydrolysis-driven gating in cystic fib... Philos Trans R Soc Lond B Biol Sci. 2009 Jan 27;364(1514):247-55. Muallem D, Vergani P
Review. ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator.
Philos Trans R Soc Lond B Biol Sci. 2009 Jan 27;364(1514):247-55., 2009-01-27 [PMID:18957373]

Abstract [show]
Proteins belonging to the ATP-binding cassette superfamily couple ATP binding and hydrolysis at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most superfamily members are transporters, while cystic fibrosis transmembrane conductance regulator (CFTR), alone, is an ion channel. Despite this functional difference, recent results have suggested that CFTR shares a common molecular mechanism with other members. ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with complete composite catalytic sites now buried at the interface. ATP hydrolysis and gamma-phosphate dissociation, with the loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where, in transporters, they drive the cycle of conformational changes that translocate the substrate across the membrane; in CFTR, they result in opening and closing (gating) of the ion-permeation pathway.

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61 For both mutants (K464A in site 1 and D1370N in site 2), opening rate was reduced at low [ATP], but fast opening could be restored by increasing [ATP] (figure 2b, open symbols). Login to comment
86 (b) Hyperbolic relationship between [ATP] and opening rates (apparent dissociation constants are 56G5, 807G185, 391G118 mM for WT (filled circles), K464A (open triangles) and D1370N (open squares), respectively). Login to comment
84 (b) Hyperbolic relationship between [ATP] and opening rates (apparent dissociation constants are 56G5, 807G185, 391G118 mM for WT (filled circles), K464A (open triangles) and D1370N (open squares), respectively). Login to comment

[hide] State-dependent modulation of CFTR gating by pyrop... J Gen Physiol. 2009 Apr;133(4):405-19. Tsai MF, Shimizu H, Sohma Y, Li M, Hwang TC
State-dependent modulation of CFTR gating by pyrophosphate.
J Gen Physiol. 2009 Apr;133(4):405-19., [PMID:19332621]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is an adenosine triphosphate (ATP)-gated chloride channel. ATP-induced dimerization of CFTR's two nucleotide-binding domains (NBDs) has been shown to reflect the channel open state, whereas hydrolysis of ATP is associated with channel closure. Pyrophosphate (PPi), like nonhydrolytic ATP analogues, is known to lock open the CFTR channel for tens of seconds when applied with ATP. Here, we demonstrate that PPi by itself opens the CFTR channel in a Mg(2+)-dependent manner long after ATP is removed from the cytoplasmic side of excised membrane patches. However, the short-lived open state (tau approximately 1.5 s) induced by MgPPi suggests that MgPPi alone does not support a stable NBD dimer configuration. Surprisingly, MgPPi elicits long-lasting opening events (tau approximately 30 s) when administrated shortly after the closure of ATP-opened channels. These results indicate the presence of two different closed states (C(1) and C(2)) upon channel closure and a state-dependent effect of MgPPi on CFTR gating. The relative amount of channels entering MgPPi-induced long-open bursts during the ATP washout phase decreases over time, indicating a time-dependent dissipation of the closed state (C(2)) that can be locked open by MgPPi. The stability of the C(2) state is enhanced when the channel is initially opened by N(6)-phenylethyl-ATP, a high affinity ATP analogue, but attenuated by W401G mutation, which likely weakens ATP binding to NBD1, suggesting that an ATP molecule remains bound to the NBD1 site in the C(2) state. Taking advantage of the slow opening rate of Y1219G-CFTR, we are able to identify a C(2)-equivalent state (C(2)*), which exists before the channel in the C(1) state is opened by ATP. This closed state responds to MgPPi much more inefficiently than the C(2) state. Finally, we show that MgAMP-PNP exerts its effects on CFTR gating via a similar mechanism as MgPPi. The structural and functional significance of our findings is discussed.

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433 Echoing this observation, the lock-open duration of MgPPi is reduced by mutations that decrease the ATP binding affinity to NBD1, K464A, and W401G, but can be partially restored by a high affinity ATP analogue, P-ATP (unpublished data; compare Powe et al., 2002; Zhou et al., 2006). Login to comment

[hide] Direct sensing of intracellular pH by the cystic f... J Biol Chem. 2009 Dec 18;284(51):35495-506. Epub . Chen JH, Cai Z, Sheppard DN
Direct sensing of intracellular pH by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel.
J Biol Chem. 2009 Dec 18;284(51):35495-506. Epub ., 2009-12-18 [PMID:19837660]

Abstract [show]
In cystic fibrosis (CF), dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel disrupts epithelial ion transport and perturbs the regulation of intracellular pH (pH(i)). CFTR modulates pH(i) through its role as an ion channel and by regulating transport proteins. However, it is unknown how CFTR senses pH(i). Here, we investigate the direct effects of pH(i) on recombinant CFTR using excised membrane patches. By altering channel gating, acidic pH(i) increased the open probability (P(o)) of wild-type CFTR, whereas alkaline pH(i) decreased P(o) and inhibited Cl(-) flow through the channel. Acidic pH(i) potentiated the MgATP dependence of wild-type CFTR by increasing MgATP affinity and enhancing channel activity, whereas alkaline pH(i) inhibited the MgATP dependence of wild-type CFTR by decreasing channel activity. Because these data suggest that pH(i) modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pH(i) dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Site 2 mutants, but not site 1 mutants, perturbed both potentiation by acidic pH(i) and inhibition by alkaline pH(i), suggesting that site 2 is a critical determinant of the pH(i) sensitivity of CFTR. The effects of pH(i) also suggest that site 2 might employ substrate-assisted catalysis to ensure that ATP hydrolysis follows NBD dimerization. We conclude that the CFTR Cl(-) channel senses directly pH(i). The direct regulation of CFTR by pH(i) has important implications for the regulation of epithelial ion transport.

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6 Because these data suggest that pHi modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pHi dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Login to comment
47 To study the CFTR variants K464A, D572N, and D1370N, we employed the vaccinia virus/bacteriophage T7 hybrid expression system to transiently express CFTR variants in HeLa cells as described previously (17, 18). Login to comment
229 At pHi 7.3, bursts of K464A-CFTR channel openings were separated by prolonged channel closures, whereas dramatically prolonged bursts of K1250M-CFTR channel openings were separated by very long-lived channel closures (Figs. 7, E and G, and 8). Login to comment
230 Although Po values of K464A-CFTR were less than those of wild-type CFTR at all pHi values tested, the effects of acidic and alkaline pHi on K464A- and wild-type CFTR were similar (Fig. 7, E and F). Login to comment
231 This suggests that K464A-CFTR does not change the pHi sensitivity of CFTR. Login to comment
246 A, C, E, and G, representative recordings show the effects of pHi on the activity of G551D-, G1349D-, K464A-, and K1250M-CFTR Cl-channels in the presence of ATP (1 mM). Login to comment
247 Dotted lines indicate where channels are closed, and downward deflections correspond to channel openings. B, D, F, and H, effects of pHi on the NPo of G551Dand G1349D-CFTR and Po of K464A- and K1250M-CFTR. Login to comment
311 Third, Hϩ ions potentiate the gating behavior of CFTR constructs bearing site-directed mutations in ATP-binding site 1 (K464A- and D572N-CFTR). Login to comment

[hide] Strict coupling between CFTR's catalytic cycle and... Proc Natl Acad Sci U S A. 2010 Jan 19;107(3):1241-6. Epub 2009 Dec 4. Csanady L, Vergani P, Gadsby DC
Strict coupling between CFTR's catalytic cycle and gating of its Cl- ion pore revealed by distributions of open channel burst durations.
Proc Natl Acad Sci U S A. 2010 Jan 19;107(3):1241-6. Epub 2009 Dec 4., 2010-01-19 [PMID:19966305]

Abstract [show]
CFTR, the ABC protein defective in cystic fibrosis, functions as an anion channel. Once phosphorylated by protein kinase A, a CFTR channel is opened and closed by events at its two cytosolic nucleotide binding domains (NBDs). Formation of a head-to-tail NBD1/NBD2 heterodimer, by ATP binding in two interfacial composite sites between conserved Walker A and B motifs of one NBD and the ABC-specific signature sequence of the other, has been proposed to trigger channel opening. ATP hydrolysis at the only catalytically competent interfacial site is suggested to then destabilize the NBD dimer and prompt channel closure. But this gating mechanism, and how tightly CFTR channel opening and closing are coupled to its catalytic cycle, remains controversial. Here we determine the distributions of open burst durations of individual CFTR channels, and use maximum likelihood to evaluate fits to equilibrium and nonequilibrium mechanisms and estimate the rate constants that govern channel closure. We examine partially and fully phosphorylated wild-type CFTR channels, and two mutant CFTR channels, each bearing a deleterious mutation in one or other composite ATP binding site. We show that the wild-type CFTR channel gating cycle is essentially irreversible and tightly coupled to the ATPase cycle, and that this coupling is completely destroyed by the NBD2 Walker B mutation D1370N but only partially disrupted by the NBD1 Walker A mutation K464A.

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No. Sentence Comment
7 We show that the wild-type CFTR channel gating cycle is essentially irreversible and tightly coupled to the ATPase cycle, and that this coupling is completely destroyed by the NBD2 Walker B mutation D1370N but only partially disrupted by the NBD1 Walker A mutation K464A. Login to comment
60 (B-D) Histograms of open burst durations for prephosphorylated WT (B), D1370N (C), and K464A (D) CFTR channels; 30-s segments of representative single-channel current recordings are shown above each panel. Login to comment
65 ATP was 2 mM for WT and D1370N, but 5 mM for K464A. Login to comment
66 Burst Duration Distribution of K464A Mutant Reveals Profoundly Altered Gating Mechanism. Login to comment
67 Although the K464A mutation lowers CFTR ATPase turnover rate ~10-fold (21), τb was essentially unaffected by this mutation (Fig. 1D Inset). Login to comment
68 However, the shape of the distribution of K464A burst durations (Fig. 1D; reconstructed from 2,327 events) clearly differed from that of WT CFTR. Login to comment
70 However, because the K464A mutant does carry out some ATP hydrolysis, albeit slowly (21), we also evaluated a partially hydrolytic mechanism by leaving k-1 in scheme 2 as a free parameter. Login to comment
73 The rate estimates from this fit suggest that in K464A CFTR, the rate of the ATP hydrolysis step (k1) is slowed by only ~4-fold compared with WT, consistent with the fact that this mutation is not in the composite NBD2 site, where ATP hydrolysis occurs. Login to comment
74 But this analysis also indicates that the K464A mutation greatly destabilizes the prehydrolytic dimer (k-1 is increased). Login to comment
84 Acceleration of Nonhydrolytic Channel Closure by the K464A Mutation Supports Microscopic Burst Duration Analysis. Login to comment
85 Assuming k-1 = 0.22 s-1 for WT, the ML fit of scheme 2 to the K464A burst distribution (Fig. 1D) suggests that rate k-1 is increased by ~15-fold in K464A CFTR. Login to comment
86 This conclusion from the distribution of microscopic burst durations is corroborated by the fact that closure of nonhydrolytic CFTR mutants and of WT channels "locked" in open bursts by nonhydrolyzable ATP analogs or by orthovanadate is greatly accelerated by the K464A mutation (16, 18, 30, 32). Login to comment
87 For instance, closure of the catalytically incompetent NBD2 Walker A mutant K1250A is accelerated ~10-fold in the double-mutant K464A/K1250A, as reported by the rate of macroscopic current decay upon ATP removal (Fig. 2B; red line is a single-exponential fit; Fig. 2C, red bar). Login to comment
97 Slow nonhydrolytic closing rate and its acceleration by the K464A mutation. Login to comment
98 (A and B) Macroscopic currents of prephosphorylated K1250A (A) and K464A/K1250A (B) CFTR channels were activated by application of 10 mM ATP. Login to comment
100 (C) Mean (±SEM) closing rates estimated as the inverses of the current relaxation time constants (τrelax), for K1250A (blue) and K464A/K1250A (red). Login to comment
104 Fig. 4 A-C compares such average parameters for fully (navy blue) and partially (royal blue) phosphorylated WT, and partially phosphorylated D1370N (green) and K464A (red) CFTR channels. Login to comment
105 Consistent with previous reports, for D1370N CFTR channels gating in near-saturating ATP (2 mM) (16), τb is ~4-fold longer than, but τib is like, that of WT (Fig. 4 B and C, green bars) (cf. refs. 9, 16, 30), whereas for prephosphorylated K464A CFTR channels in saturating ATP (5 mM) (16), τb is comparable to, but τib is at least ~2-fold longer than, that of WT (Fig. 4 B and C, red bars) (9, 14, 16 but cf. ref. 32). Login to comment
115 However, the fit for K464A provides additional support for this assignment. Login to comment
117 Thus, the set k1' = 15.4 s-1 , k2' = 4.30 s-1 , k-1' = 3.39 s-1 fits the observed pdf for K464A just as well as the set displayed in Fig. 4E (k1 = 0.91 s-1 , k2 = 18.8 s-1 , k-1 = 3.39 s-1 ). Login to comment
118 But this alternative set can be ruled out as it would yield an essentially hydrolytic mechanism for K464A (with a coupling ratio of ~82%) in contradiction of the observed severe defect in ATPase turnover rate (21). Login to comment
120 The NBD1 Walker A mutant K464A has received much previous attention. Login to comment
121 That this mutation, in a catalytically inactive binding site (17, 18), affects channel gating only slightly (Fig. 4D; cf. refs. 16, 21, 32) contrasts with its substantial suppression of the rate of ATP hydrolysis of purified K464A CFTR protein (to <10% of WT) (21). Login to comment
122 The altered shape of the distribution of K464A burst durations (Fig. 1D) now provides a satisfying explanation for this dissociation between channel cycle time and ATPase rate. Login to comment
123 The ratio k1/(k1 + k-1) estimated from the scheme 2 fit (Fig.4E Left and Right, red bars) suggests that in K464A approximately one out of every five bursts proceeds through the normal irreversible hydrolytic pathway (Fig. 4F). Login to comment
124 This "coupling ratio" between channel opening and ATP hydrolysis of ~21% for K464A CFTR contrasts with that of ≥95% for WT channels (blue bars, Fig. 4E Left and Right and Fig. 4F). Login to comment
125 Because the cycle time of K464A channels is prolonged ~2-fold compared with WT (Fig. 4D), the predicted overall ATPase turnover rate is on the order of 10% of WT, which is in very reasonable agreement with the measured value (21). Login to comment
161 (A-D) Open probabilities (A), mean burst (B), and interburst (C) durations obtained from multichannel fits, and calculated channel cycle times (D) for fully (navy blue) and partially (royal blue) phosphorylated WT, and partially phosphorylated D1370N (green) and K464A (red) CFTR. Login to comment
162 [ATP] was 2 mM for WT and D1370N, but 5 mM for K464A. Login to comment
164 (E) ML estimates of rates k1 (Left), k2 (Center), and k-1 (Right) for fully (navy blue) and partially (royal blue) phosphorylated WT, and partially phosphorylated D1370N (green) and K464A (red) CFTR channels. Login to comment
169 Probabilities for exiting state O1 (Top Right) in either of two possible directions are printed in color for partially phosphorylated WT (blue), K464A (red), and D1370N (green). Login to comment
184 Therefore, although both methods yielded qualitatively similar results, we used the distributions obtained using method (i) for WT and K464A (Figs. 1 B and D, 3B, and 4), and that obtained using method (ii) for D1370N (Fig. 1C). Login to comment

[hide] The cystic fibrosis-causing mutation deltaF508 aff... J Biol Chem. 2010 Nov 12;285(46):35825-35. Epub 2010 Jul 28. Thibodeau PH, Richardson JM 3rd, Wang W, Millen L, Watson J, Mendoza JL, Du K, Fischman S, Senderowitz H, Lukacs GL, Kirk K, Thomas PJ
The cystic fibrosis-causing mutation deltaF508 affects multiple steps in cystic fibrosis transmembrane conductance regulator biogenesis.
J Biol Chem. 2010 Nov 12;285(46):35825-35. Epub 2010 Jul 28., 2010-11-12 [PMID:20667826]

Abstract [show]
The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the DeltaF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the DeltaF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that DeltaF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.

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200 Mutations of the Walker A lysine (K464A and K1250A in NBD1 and NBD2, respectively) have been shown to dramatically decrease ATP affinity (40). Login to comment
206 The NBD1 K464A mutation also failed to rescue ⌬F508 trafficking. Login to comment
207 However, when introduced into the wild type background, the K464A reduced CFTR maturation, as evidenced by a decrease in band C. Login to comment
280 B, mutation of the composite ATP-binding site in NBD1, K464A, adversely affects the trafficking of wild type CFTR. Login to comment
330 In contrast, the K464A NBD1 ATP-binding mutant decreased wild type CFTR maturation. Login to comment

[hide] Ligand-driven vectorial folding of ribosome-bound ... Mol Cell. 2011 Mar 18;41(6):682-92. Khushoo A, Yang Z, Johnson AE, Skach WR
Ligand-driven vectorial folding of ribosome-bound human CFTR NBD1.
Mol Cell. 2011 Mar 18;41(6):682-92., 2011-03-18 [PMID:21419343]

Abstract [show]
The mechanism by which protein folding is coupled to biosynthesis is a critical, but poorly understood, aspect of protein conformational diseases. Here we use fluorescence resonance energy transfer (FRET) to characterize tertiary structural transitions of nascent polypeptides and show that the first nucleotide-binding domain (NBD1) of human CFTR, whose folding is defective in cystic fibrosis, folds via a cotranslational multistep pathway as it is synthesized on the ribosome. Folding begins abruptly as NBD1 residues 389-500 emerge from the ribosome exit tunnel, initiating compaction of a small, N-terminal alpha/beta-subdomain. Real-time kinetics of synchronized nascent chains revealed that subdomain folding is rapid, occurs coincident with synthesis, and is facilitated by direct ATP binding to the nascent polypeptide. These findings localize the major CF defect late in the NBD1 folding pathway and establish a paradigm wherein a cellular ligand promotes vectorial domain folding by facilitating an energetically favored local peptide conformation.

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114 In addition, mutations that inhibit ATP binding to NBD1 in full-length CFTR (W401A, A462F, and K464A [Berger et al., 2005]) also inhibited binding to truncated NBD1 (Figures 5E and 5F). Login to comment
205 The CFP-NBD1 ATP-binding mutant contained W401A, A462F, and K464A mutations in the ATP-binding site as predicted by the crystal structure (2BBO). Login to comment

[hide] Mutant cycles at CFTR's non-canonical ATP-binding ... J Gen Physiol. 2011 Jun;137(6):549-62. doi: 10.1085/jgp.201110608. Epub 2011 May 16. Szollosi A, Muallem DR, Csanady L, Vergani P
Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating.
J Gen Physiol. 2011 Jun;137(6):549-62. doi: 10.1085/jgp.201110608. Epub 2011 May 16., [PMID:21576373]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460-1348 and 460-1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

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59 A mutation in the conserved Walker A lysine of NBD1, K464A, reduced channel opening rate at low [ATP] (Vergani et al., 2003), suggesting that ATP binding at site 1 can be made rate limiting for channel opening. Login to comment
61 In other studies, neither the K464A mutation (Powe et al., 2002) nor mutation of an aromatic residue (W401), which is seen to stack against the adenine moiety of ATP in some crystals of CFTR NBD1 (Lewis et al., 2005, but cf. Login to comment

[hide] Structure and function of the CFTR chloride channe... Physiol Rev. 1999 Jan;79(1 Suppl):S23-45. Sheppard DN, Welsh MJ
Structure and function of the CFTR chloride channel.
Physiol Rev. 1999 Jan;79(1 Suppl):S23-45., [PMID:9922375]

Abstract [show]
Structure and Function of the CFTR Chloride Channel. Physiol. Rev. 79, Suppl.: S23-S45, 1999. - The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ABC transporter family that forms a novel Cl- channel. It is located predominantly in the apical membrane of epithelia where it mediates transepithelial salt and liquid movement. Dysfunction of CFTR causes the genetic disease cystic fibrosis. The CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. Here we review the structure and function of this unique channel, with a focus on how the various domains contribute to channel function. The MSDs form the channel pore, phosphorylation of the R domain determines channel activity, and ATP hydrolysis by the NBDs controls channel gating. Current knowledge of CFTR structure and function may help us understand better its mechanism of action, its role in electrolyte transport, its dysfunction in cystic fibrosis, and its relationship to other ABC transporters.

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375 This is consistent with the finding that in the absence predicted to decrease the rate of hydrolysis at NBD1, for example, K464A and Q552A, would thus increase the in-of ATP, the channel does not open. Login to comment

[hide] Control of CFTR channel gating by phosphorylation ... Physiol Rev. 1999 Jan;79(1 Suppl):S77-S107. Gadsby DC, Nairn AC
Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis.
Physiol Rev. 1999 Jan;79(1 Suppl):S77-S107., [PMID:9922377]

Abstract [show]
Control of CTFR Channel Gating by Phosphorylation and Nucleotide Hydrolysis. Physiol. Rev. 79, Suppl.: S77-S107, 1999. - The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is the protein product of the gene defective in cystic fibrosis, the most common lethal genetic disease among Caucasians. Unlike any other known ion channel, CFTR belongs to the ATP-binding cassette superfamily of transporters and, like all other family members, CFTR includes two cytoplasmic nucleotide-binding domains (NBDs), both of which bind and hydrolyze ATP. It appears that in a single open-close gating cycle, an individual CFTR channel hydrolyzes one ATP molecule at the NH2-terminal NBD to open the channel, and then binds and hydrolyzes a second ATP molecule at the COOH-terminal NBD to close the channel. This complex coordinated behavior of the two NBDs is orchestrated by multiple protein kinase A-dependent phosphorylation events, at least some of which occur within the third large cytoplasmic domain, called the regulatory domain. Two or more kinds of protein phosphatases selectively dephosphorylate distinct sites. Under appropriately controlled conditions of progressive phosphorylation or dephosphorylation, three functionally different phosphoforms of a single CFTR channel can be distinguished on the basis of channel opening and closing kinetics. Recording single CFTR channel currents affords an unprecedented opportunity to reproducibly examine, and manipulate, individual ATP hydrolysis cycles in a single molecule, in its natural environment, in real time.

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558 First,be sequential, although the precise nature and mechanism of the ordering remain to be determined. In keeping with they noted that the opening rate of mutant K464A CFTR channels was only approximately twofold lower than thatthe proposed role of PKA-mediated phosphorylation, presumably largely within the R domain, in modulating the of wild-type CFTR, whereas the equivalent mutation in other nucleoside triphosphatases (NTPases) reduces thefunction of NBD2, it has recently been shown that neither AMP-PNP nor PPi (both of which are believed to bind hydrolysis rate by several orders of magnitude. Login to comment
560 Recent analyses of the dependence on temperature K464A in purified CFTR slows ATP hydrolysis only less than twofold (159). Login to comment

[hide] Walker mutations reveal loose relationship between... Biochemistry. 1999 Feb 2;38(5):1463-8. Ramjeesingh M, Li C, Garami E, Huan LJ, Galley K, Wang Y, Bear CE
Walker mutations reveal loose relationship between catalytic and channel-gating activities of purified CFTR (cystic fibrosis transmembrane conductance regulator).
Biochemistry. 1999 Feb 2;38(5):1463-8., 1999-02-02 [PMID:9931011]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ATPase and as a chloride channel. It has been hypothesized, on the basis of electrophysiological findings, that the catalytic activity of CFTR is tightly coupled to the opening and closing of the channel gate. In the present study, to determine the structural basis for the ATPase activity of CFTR, we assessed the effect of mutations within the "Walker A" consensus motifs on ATP hydrolysis by the purified, intact protein. Mutation of the lysine residue in the "Walker A" motif of either the first nucleotide binding fold (CFTRK464A) or the second nucleotide binding fold (CFTRK1250A) inhibited the ATPase activity of the purified intact CFTR protein significantly, by greater than 50%. This finding suggests that the two nucleotide binding folds of CFTR are functioning cooperatively in catalysis. However, the rate of channel gating was only significantly inhibited in one of these purified mutants, CFTRK1250A, suggesting that ATPase activity may not be tightly coupled to channel gating as previously hypothesized.

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21 While it was determined that the K464A mutation abrogated ATPase activity of the CFTR-NBF1 fusion protein (15), the effect of the K1250A mutation in the context of the CFTR-NBF2 fusion protein is not known. Login to comment
32 A small cassette containing the specific mutation, i.e., a BspE1/SphI fragment for the K464A mutation or a Pm1I/Tth111I fragment for the K1250A mutation was subcloned into a new pBQ6.2 and then sequenced to confirm the introduction of the mutations. Login to comment
33 Ultimately, K464A (as an XbaI/SphI fragment) or K1250A (as a Pm1I/Tth111I fragment) was subcloned into pBlueBac4 (Invitrogen, Carlsbad, CA) for baculovirus expression. Login to comment
123 Disruption of the Chloride Channel ActiVity of the Intact Purified CFTR Protein by Mutation of the Walker A Lysine in either NBF1 or NBF2. We assessed the consequences of each of the Walker lysine mutations, K464A and K1250A, on the chloride channel activity of CFTR using two assays. Login to comment

[hide] Mutations in either nucleotide-binding site of P-g... Biochemistry. 1998 Mar 31;37(13):4592-602. Urbatsch IL, Beaudet L, Carrier I, Gros P
Mutations in either nucleotide-binding site of P-glycoprotein (Mdr3) prevent vanadate trapping of nucleotide at both sites.
Biochemistry. 1998 Mar 31;37(13):4592-602., 1998-03-31 [PMID:9521779]

Abstract [show]
Vanadate trapping of nucleotide and site-directed mutagenesis were used to investigate the role of the two nucleotide-binding (NB) sites in the regulation of ATP hydrolysis by P-glycoprotein (mouse Mdr3). Mdr3, tagged with a hexahistidine tail, was overexpressed in the yeast Pichia pastoris and purified to about 90% homogeneity by Ni-affinity chromatography. This protocol yielded purified, reconstituted Mdr3 which exhibited high verapamil stimulation of ATPase activity with a Vmax of 4.2 micromol min-1 mg-1 and a KM of 0.7 mM, suggesting that Mdr3 purified from P. pastoris is highly functional. Point mutations were introduced into the core consensus sequence of the Walker A or B motifs in each of the two NB sites. The mutants K429R, K1072R (Walker A) and D551N, D1196N (Walker B) were functionally impaired and unable to confer cellular resistance to the fungicide FK506 in the yeast Saccharomyces cerevisiae. Single and double mutants (K429R/K1072R, D551N/D1196N) were expressed in P. pastoris, and the effect of these mutations on the ATPase activity of Mdr3 was characterized. Purified reconstituted Mdr3 mutants showed no detectable ATPase activity compared to proteoliposomes purified from negative controls (<5% of wild-type Mdr3). Vanadate readily induced trapping of 8-azido-nucleotide in the wild-type enzyme after a short 10 s incubation, and specific photolabeling of Mdr3 after UV irradiation. No such vanadate-induced trapping/photolabeling was observed in any of the mutants, even after a 60 min trapping period at 37 degrees C. Since vanadate trapping with 8-azido-ATP requires hydrolysis of the nucleotide, the data suggest that 8-azido-ATP hydrolysis is dramatically impaired in all of the mutant proteins (<0.3% activity). These results show that mutations in either NB site prevent single turnover and vanadate trapping of nucleotide in the nonmutant site. These results further suggest that the two NB sites cannot function independently as catalytic sites in the intact molecule. In addition, the N- or C-terminal NB sites appear functionally indistinguishable, and cooperative interactions absolutely required for ATP hydrolysis may originate from both sites.

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254 In the cystic fibrosis transmembrane conductance regulator (CFTR), mutations of the conserved Walker A lysine altered the conductive properties of the Cl- channel: the K464A mutation in NB1 decreased the frequency of channel openings, whereas K1250A or K1250M in NB2 prolonged the open state of the channel (59). Login to comment

[hide] Alternating access to the transmembrane domain of ... J Biol Chem. 2012 Mar 23;287(13):10156-65. Epub 2012 Feb 1. Wang W, Linsdell P
Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7).
J Biol Chem. 2012 Mar 23;287(13):10156-65. Epub 2012 Feb 1., [PMID:22303012]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.

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52 These two reporter cysteine substitutions were combined with mutations in the NBDs that affect ATP-dependent channel gating: K464A (NBD1) and E1371Q (NBD2). Login to comment
53 As discussed in detail in our recent study (22), these NBD mutations are expected either to decrease (K464A) or increase (E1371Q) overall CFTR channel activity via well characterized effects on ATP-dependent channel gating. Login to comment
77 Specifically, following the lead of other groups (25, 26), we used the NBD1 mutation K464A to decrease overall channel activity, and the NBD2 mutation E1371Q to increase channel activity. Login to comment
78 Previously we showed that these mutations mimic the effects of decreasing channel activity with low cytoplasmic ATP concentrations (K464A) or by "locking" channels in the open state by treatment with 2 mM sodium pyrophosphate (E1371Q) (22). Login to comment
88 In both cases, the rate of modification by MTSES was significantly decreased by the K464A mutation and significantly increased by the E1371Q mutation, effects that are quantified in Fig. 1C. Login to comment
89 For modification of T338C, the mean modification rate constant was decreased ϳ2.4-fold in a K464A background and increased ϳ3.9-fold in E1371Q, whereas the modification rate constant for L102C was decreased by ϳ26% in K464A and increased ϳ2.0-fold in E1371Q. Login to comment
93 As shown in Fig. 2, application of a low concentration of Au(CN)2 - (2 ␮M) caused a rapid inhibition of current carried by both T338C and L102C. Login to comment
94 As with MTSES, the rate of modification by Au(CN)2 - was significantly decreased by the K464A mutation (by ϳ1.8-fold for T338C and ϳ3.4-fold for L102C) and significantly increased by the E1371Q mutation (by ϳ5.6-fold for T338C and ϳ1.8-fold for L102C), as well as by pyrophosphate treatment (by ϳ6.0-fold for T338C and ϳ2.0-fold for L102C; data not shown). Login to comment
99 Expression of all E1371Q-CFTR constructs led to the appearance of constitutive, cAMP-insensitive but GlyH-101-inhibited whole cell currents (supplemental Fig. S2). Login to comment
104 Fig. 4A shows examples of the rate of current inhibition in response to application of a common concentration of MTSES (1 ␮M) in T338C, T338C/K464A, and T338C/E1371Q. Login to comment
109 The decline in current amplitude following Au(CN)2 - application has been fitted by a single exponential function. C, average modification rate constants(k)forAu(CN)2 - ,calculatedfromfitstodatasuchasthoseshowninAandB.AsterisksindicateasignificantdifferencefromthecysteinemutantsT338C and L102C (black bars) (p Ͻ 0.02). Login to comment
110 Data are mean from three or four patches. Alternating Access Model of CFTR MARCH 23, 2012ȂVOLUME 287•NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 10159 fication (Fig. 4B) suggests an increase of ó3.7-fold in T338C/ K464A and a dramatic decrease of ϳ35-fold in T338C/E1371Q compared with T338C alone. Login to comment
114 Quantification of the rate constant for modification (Fig. 5C) suggests, for modification of T338C, an increase of ϳ5.7-fold in K464A and a decrease of ϳ150-fold in E1371Q, and for modification of L102C, an increase of ϳ2.3-fold in K464A and a decrease of ϳ2.7-fold in E1371Q. Login to comment
116 Changing Patterns of Accessibility Suggest Marker Cysteine Residues "Switch Sides" of Membrane during Gating-The effects of NBD mutations on the rate of modification of T338C and L102C by internal cysteine-reactive reagents (estimated from experiments on inside-out membrane patches) and by external cysteine-reactive reagents (estimated from whole cell current recording experiments) are compared in Fig. 6. Login to comment
120 In each panel, it can be seen that the rate of modification by internal MTSES and Au(CN)2 - increases in the order K464A Ͻ Cys-less Ͻ E1371Q, whereas modification by extracellular MTSES (in T338C) and Au(CN)2 - shows the opposite pattern, K464A Ͼ Cys-less Ͼ E1371Q. Login to comment
123 Given the known effects of the K464A and E1371Q mutations on ATP-dependent channel gating, it seems reasonable to us to infer that the factor causing this movement from one side of the membrane to the other is channel gating: when the channel is closed (enriched in the K464A constructs), accessibility of these cysteines from the outside is increased, and when the channel is open (enriched in the E1371Q constructs), their accessibility from the inside is increased. Login to comment
178 Each panel illustrates the change in modification rate constant for the same reporter cysteine (T338C in A and C, L102C in B and D) in three different backgrounds (K464A, Cys-less, and E1371Q), for modification by MTSES (A and B) or Au(CN)2 - (C and D) applied to the intracellular (●, inside) or extracellular (E, outside) side of the membrane. Login to comment
54 These two reporter cysteine substitutions were combined with mutations in the NBDs that affect ATP-dependent channel gating: K464A (NBD1) and E1371Q (NBD2). Login to comment
55 As discussed in detail in our recent study (22), these NBD mutations are expected either to decrease (K464A) or increase (E1371Q) overall CFTR channel activity via well characterized effects on ATP-dependent channel gating. Login to comment
80 Specifically, following the lead of other groups (25, 26), we used the NBD1 mutation K464A to decrease overall channel activity, and the NBD2 mutation E1371Q to increase channel activity. Login to comment
81 Previously we showed that these mutations mimic the effects of decreasing channel activity with low cytoplasmic ATP concentrations (K464A) or by "locking" channels in the open state by treatment with 2 mM sodium pyrophosphate (E1371Q) (22). Login to comment
126 In each panel, it can be seen that the rate of modification by internal MTSES and Au(CN)2 afa; increases in the order K464A b0d; Cys-less b0d; E1371Q, whereas modification by extracellular MTSES (in T338C) and Au(CN)2 afa; shows the opposite pattern, K464A b0e; Cys-less b0e; E1371Q. Login to comment
129 Given the known effects of the K464A and E1371Q mutations on ATP-dependent channel gating, it seems reasonable to us to infer that the factor causing this movement from one side of the membrane to the other is channel gating: when the channel is closed (enriched in the K464A constructs), accessibility of these cysteines from the outside is increased, and when the channel is open (enriched in the E1371Q constructs), their accessibility from the inside is increased. Login to comment
185 Each panel illustrates the change in modification rate constant for the same reporter cysteine (T338C in A and C, L102C in B and D) in three different backgrounds (K464A, Cys-less, and E1371Q), for modification by MTSES (A and B) or Au(CN)2 afa; (C and D) applied to the intracellular (cf;, inside) or extracellular (E, outside) side of the membrane. Login to comment

[hide] Conformational change opening the CFTR chloride ch... Biochim Biophys Acta. 2012 Mar;1818(3):851-60. Epub 2012 Jan 2. Wang W, Linsdell P
Conformational change opening the CFTR chloride channel pore coupled to ATP-dependent gating.
Biochim Biophys Acta. 2012 Mar;1818(3):851-60. Epub 2012 Jan 2., [PMID:22234285]

Abstract [show]
Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are controlled by ATP binding and hydrolysis by its nucleotide binding domains (NBDs). This is presumed to control opening of a single "gate" within the permeation pathway, however, the location of such a gate has not been described. We used patch clamp recording to monitor access of cytosolic cysteine reactive reagents to cysteines introduced into different transmembrane (TM) regions in a cysteine-less form of CFTR. The rate of modification of Q98C (TM1) and I344C (TM6) by both [2-sulfonatoethyl] methanethiosulfonate (MTSES) and permeant Au(CN)(2)(-) ions was reduced when ATP concentration was reduced from 1mM to 10muM, and modification by MTSES was accelerated when 2mM pyrophosphate was applied to prevent channel closure. Modification of K95C (TM1) and V345C (TM6) was not affected by these manoeuvres. We also manipulated gating by introducing the mutations K464A (in NBD1) and E1371Q (in NBD2). The rate of modification of Q98C and I344C by both MTSES and Au(CN)(2)(-) was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected. These results suggest that access from the cytoplasm to K95 and V345 is similar in open and closed channels. In contrast, modifying ATP-dependent channel gating alters access to Q98 and I344, located further into the pore. We propose that ATP-dependent gating of CFTR is associated with the opening and closing of a gate within the permeation pathway at the level of these pore-lining amino acids.

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5 We also manipulated gating by introducing the mutations K464A (in NBD1) and E1371Q (in NBD2). Login to comment
6 The rate of modification of Q98C and I344C by both MTSES and Au(CN)2 - was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected. Login to comment
54 In (D) channels also had the NBD1 mutation K464A, and in (E) channels also had the NBD2 mutation E1371Q. Login to comment
60 Additional mutations were introduced into the cys-less background using the QuikChange site-directed mutagenesis -200 -150 -100 -50 0 I (pA) Time (s) K95C A) 1 mM ATP -180 -120 -60 0 Q98C -400 -300 -200 -100 0 -200 -150 -100 -50 0 I344C -300 -200 -100 0 -500 -400 -300 -200 -100 0 V345C -250 -200 -150 -100 -50 0 -300 -200 -100 0 -600 -400 -200 0 -750 -500 -250 0 -600 -400 -200 0 -800 -600 -400 -200 0 I (pA) Time (s) 20 µM MTSES 20 µM MTSES 20 µM MTSES200 µM MTSES C) 1 mM ATP + 2 mM PPi E) E1371Q (1 mM ATP) I (pA) Time (s) D) K464A (1 mM ATP) B) 10 µM ATP -100 -75 -50 -25 0 -200 -150 -100 -50 0 -80 -60 -40 -20 0 -80 -60 -40 -20 0 -120 -90 -60 -30 0 -80 -60 -40 -20 0 -60 -40 -20 0 -300 -200 -100 0 I (pA) Time (s) I (pA) Time (s) 0 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 system (Agilent Technologies, Santa Clara, CA, USA) and verified by DNA sequencing. Login to comment
95 In (B), NBD function has been altered by the mutations K464A (NBD1) and E1371Q (NBD2). Login to comment
105 Indeed, previous studies of pore accessibility changes have taken advantage of NBD mutations K464A and E1371Q [10,11]. Login to comment
106 The NBD1 mutation K464A decreases channel opening rate and has been described as slightly decreasing overall open probability at moderate ATP concentrations [9,28,29], whereas mutagenesis of the key catalytic glutamate residue E1371 in NBD2 prevents ATP hydrolysis to cause dramatic prolongation of CFTR channel open times, presumably leading to an increase in open probability [1,30,31]. Login to comment
107 To alter channel gating by non-pharmacological means, we therefore combined the NBD mutations K464A and E1371Q with each of the four cysteine mutants described above. Login to comment
111 The K464A mutation significantly decreased the rate of MTSES modification at Q98C and I344C (2.5-2.9-fold decrease in modification rate constant; Pb0.005) but had no effect on the rate of modification at K95C or V345C (P>0.5) (Fig. 3B). Login to comment
150 We therefore compared the rate of Au(CN)2 - inhibition in K95C, Q98C and I344C at two different ATP concentrations (10 μM and 1 mM), as well as in channels also bearing the NBD mutations K464A or E1371Q (Fig. 6). Login to comment
151 Quantification of the mean modification rate constant demonstrated that decreasing ATP -300 -200 -100 0 -400 -300 -200 -100 0 -200 -150 -100 -50 0 -60 -40 -20 0 -120 -80 -40 0 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 -100 -75 -50 -25 0 -90 -60 -30 0 -90 -60 -30 0 -120 -80 -40 0 A) 1 mM ATP I (pA) I (pA) I (pA) Time (s) 200 nM Au(CN)2 C) K464A (1 mM ATP) D) E1371Q (1 mM ATP) 2 µM Au(CN)2 200 nM Au(CN)2 I (pA) Time (s) Time (s) -200 -150 -100 -50 0 -90 -60 -30 0 -120 -80 -40 0 K95C Q98C I344C B) 10 µM ATP Time (s) Fig. 6. Timecourse of modification by Au(CN)2 - . Login to comment
153 Reporter cysteines (K95C, Q98C, and I344C as indicated) were examined in isolation (A, B) or combined with the NBD mutations K464A (C) or E1371Q (D). Login to comment
157 In addition, the K464A mutation significantly decreased the rate of Au(CN)2 - modification of Q98C and I344C (1.4-1.5-fold decrease in modification rate constant with 1 mM ATP; Pb0.005) but had no effect on the rate of modification at K95C (P>0.5) (Fig. 7). Login to comment
167 The rate of modification at these two sites was significantly decreased by lowering ATP concentration (Figs. 3A, 7) and by the K464A mutation (Figs. 3B, 7), and significantly increased by PPi (Fig. 3A) and the E1371Q mutation (Figs. 3B, 7). Login to comment
180 For example, reducing ATP concentration to 10 μM is expected to decrease open probability around tenfold [25], whereas the K464A mutation is usually reported as having only a minor effect on open probability [9,28,29]. Login to comment
181 Perhaps surprisingly, then, we find the effects of low ATP and the K464A mutation on modification rate constants for Q98C and I344C to be quantitatively similar and in the range of 1.5 to 3-fold (Figs. 3, 7). Login to comment

[hide] Fluoride stimulates cystic fibrosis transmembrane ... Am J Physiol. 1998 Mar;274(3 Pt 1):L305-12. Berger HA, Travis SM, Welsh MJ
Fluoride stimulates cystic fibrosis transmembrane conductance regulator Cl- channel activity.
Am J Physiol. 1998 Mar;274(3 Pt 1):L305-12., [PMID:9530164]

Abstract [show]
While studying the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR), we found that addition of F- to the cytosolic surface of excised, inside-out membrane patches reversibly increased Cl- current in a dose-dependent manner. Stimulation required prior phosphorylation and the presence of ATP. F- increased current even in the presence of deferoxamine, which chelates Al3+, suggesting that stimulation was not due to AlF4-. F- also stimulated current in a CFTR variant that lacked a large part of the R domain, suggesting that the effect was not mediated via this domain. Studies of single channels showed that F- increased the open-state probability by slowing channel closure from bursts of activity; the mean closed time between bursts and single-channel conductance was not altered. These results suggested that F- influenced regulation by the cytosolic domains, most likely the nucleotide-binding domains (NBDs). Consistent with this, we found that mutation of a conserved Walker lysine in NBD2 changed the relative stimulatory effect of F- compared with wild-type CFTR, whereas mutation of the Walker lysine in NBD1 had no effect. Based on these and previous data, we speculate that F- interacts with CFTR, possibly via NBD2, and slows the rate of channel closure.

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230 Effect of increasing F- concentration on current in wild-type CFTR, CFTR-K464A, and CFTR-K1250M. Login to comment
233 *P Ͻ 0.005 compared with wild-type CFTR and *P Ͻ 0.026 compared with CFTR-K464A. Login to comment
222 Effect of increasing F2 concentration on current in wild-type CFTR, CFTR-K464A, and CFTR-K1250M. Login to comment
225 *P , 0.005 compared with wild-type CFTR and *P , 0.026 compared with CFTR-K464A. Login to comment

[hide] CFTR Cl- channel and CFTR-associated ATP channel: ... EMBO J. 1998 Feb 16;17(4):898-908. Sugita M, Yue Y, Foskett JK
CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates.
EMBO J. 1998 Feb 16;17(4):898-908., [PMID:9463368]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is regulated by phosphorylation of the R domain and ATP hydrolysis at two nucleotide-binding domains (NBDs). It is controversial whether CFTR conducts ATP or whether CFTR might be closely associated with a separate ATP conductance. To characterize ATP channels associated with CFTR, we analyzed Cl- and ATP single channel-currents in excised inside-out membrane patches from MDCK epithelial cells transiently expressing CFTR. With 100 mM ATP in the pipette and 140 mM Cl- in the bath, ATP channels were associated with CFTR Cl- channels in two-thirds of patches that included CFTR. CFTR Cl- channels and CFTR-associated ATP channels had slope conductances of 7.4 pS and 5.2 pS, respectively, and had distinct reversal potentials and sensitivities to channel blockers. CFTR-associated ATP channels exhibited slow gating kinetics that depended on the presence of protein kinase A and cytoplasmic ATP, similar to CFTR Cl- channels. Gating kinetics of the ATP channels as well as the CFTR Cl- channels were similarly affected by non-hydrolyzable ATP analogues and mutations in the CFTR R domain and NBDs. Our results indicate that phosphorylation- and nucleotide-hydrolysis-dependent gating of CFTR is directly involved in gating of an associated ATP channel. However, the permeation pathways for Cl- and ATP are distinct and the ATP conduction pathway is not obligatorily associated with the expression of CFTR.

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131 The residues altered in CFTR∆R-S660A, CFTR S-oct-D, K464A and K1250A mutants are shown. Login to comment
155 These NBD1 and NBD2 mutants, containing the individual mutations K464A and K1250A, respectively, were expressed in MDCK cells and single-channel currents of CFTR Cl- channels and CFTR-associated ATP channels were analyzed. Login to comment
161 the K464A mutant exhibited no significant differences in Po (p Ͼ0.05), to (p Ͼ0.05) and the mean closed time (tc) (p Ͼ0.15), although there was a tendency towards increased tc and shorter to (Figure 10B, C, D and E). Login to comment
163 The K464A mutation similarly had no significant effects on the gating of the CFTR-associated ATP channels (Figure 10B, C, D and E). Login to comment
173 (B) Current traces from a MDCK cell expressing K464A at various membrane potentials (representative of nine independently observed channels). Login to comment
205 Mutations in the conserved Walker A motif lysines of NBD1 (K464A) and NBD2 (K1250A) are thought to attenuate ATP hydrolysis with minimal effect on ATP binding (Sung et al., 1988; Schneider et al., 1994; Carson et al., 1995). Login to comment
206 Previous analyses of the K464A mutant indicated that it had a reduced Po due to an increased closed time, although these effects were not pronounced (Carson et al., 1995; Gunderson and Kopito, 1995). Login to comment
272 Acknowledgements We thank M.Welsh for providing the CFTR∆R-S660A and CFTR S-oct-D mutants, R.Kopito for providing the K1250A and K464A mutants, J.Engelhardt for providing the R347E mutant, U.Patel for her precious technical help and D.Mak for helpful discussions. Login to comment
138 The residues altered in CFTRƊR-S660A, CFTR S-oct-D, K464A and K1250A mutants are shown. Login to comment
170 the K464A mutant exhibited no significant differences in Po (p b0e;0.05), to (p b0e;0.05) and the mean closed time (tc) (p b0e;0.15), although there was a tendency towards increased tc and shorter to (Figure 10B, C, D and E). Login to comment
172 The K464A mutation similarly had no significant effects on the gating of the CFTR-associated ATP channels (Figure 10B, C, D and E). Login to comment
183 (B) Current traces from a MDCK cell expressing K464A at various membrane potentials (representative of nine independently observed channels). Login to comment
215 Mutations in the conserved Walker A motif lysines of NBD1 (K464A) and NBD2 (K1250A) are thought to attenuate ATP hydrolysis with minimal effect on ATP binding (Sung et al., 1988; Schneider et al., 1994; Carson et al., 1995). Login to comment
216 Previous analyses of the K464A mutant indicated that it had a reduced Po due to an increased closed time, although these effects were not pronounced (Carson et al., 1995; Gunderson and Kopito, 1995). Login to comment
282 Acknowledgements We thank M.Welsh for providing the CFTRƊR-S660A and CFTR S-oct-D mutants, R.Kopito for providing the K1250A and K464A mutants, J.Engelhardt for providing the R347E mutant, U.Patel for her precious technical help and D.Mak for helpful discussions. Login to comment

[hide] ClC and CFTR chloride channel gating. Annu Rev Physiol. 1998;60:689-717. Foskett JK
ClC and CFTR chloride channel gating.
Annu Rev Physiol. 1998;60:689-717., [PMID:9558482]

Abstract [show]
Chloride channels are widely expressed and play important roles in cell volume regulation, transepithelial transport, intracellular pH regulation, and membrane excitability. Most chloride channels have yet to be identified at a molecular level. The ClC gene family and the cystic fibrosis transmembrane conductance regulator (CFTR) are distinct chloride channels expressed in many cell types, and mutations in their genes are the cause of several diseases including myotonias, cystic fibrosis, and kidney stones. Because of their molecular definition and roles in disease, these channels have been studied intensively over the past several years. The focus of this review is on recent studies that have provided new insights into the mechanisms governing the opening and closing, i.e. gating, of the ClC and CFTR chloride channels.

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307 In one study, ATP hydrolysis by a recombinant NBD1-maltose-binding protein fusion protein was inhibited by replacement of the Walker A lysine with alanine (K464A) (179), confirming predictions. Login to comment
308 K464A CFTR channels have reduced Po (135, 145). Login to comment
314 Activation of D572 and G551 mutants is inhibited (153, 154), and channel gating of Q552 mutants is altered (138) similarly to those for the K464A mutant. Login to comment

[hide] CFTR: domains, structure, and function. J Bioenerg Biomembr. 1997 Oct;29(5):443-51. Devidas S, Guggino WB
CFTR: domains, structure, and function.
J Bioenerg Biomembr. 1997 Oct;29(5):443-51., [PMID:9511929]

Abstract [show]
Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) (Collins, 1992). Over 500 naturally occurring mutations have been identified in CF gene which are located in all of the domains of the protein (Kerem et al., 1990; Mercier et al., 1993; Ghanem et al., 1994; Fanen et al., 1992; Ferec et al., 1992; Cutting et al., 1990). Early studies by several investigators characterized CFTR as a chloride channel (Anderson et al.; 1991b,c; Bear et al., 1991). The complex secondary structure of the protein suggested that CFTR might possess other functions in addition to being a chloride channel. Studies have established that the CFTR functions not only as a chloride channel but is indeed a regulator of sodium channels (Stutts et al., 1995), outwardly rectifying chloride channels (ORCC) (Gray et al., 1989; Garber et al., 1992; Egan et al., 1992; Hwang et al., 1989; Schwiebert et al., 1995) and also the transport of ATP (Schwiebert et al., 1995; Reisin et al., 1994). This mini-review deals with the studies which elucidate the functions of the various domains of CFTR, namely the transmembrane domains, TMD1 and TMD2, the two cytoplasmic nucleotide binding domains, NBD1 and NBD2, and the regulatory, R, domain.

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113 (1995) demonstrated that CFTR variants which contained mutations in the conserved Walker A motif of either NBD1 (K464A) or NBD2 (K1250M and K1250A) decreased the open probability of the channel compared to wt-CFTR. Login to comment
114 Mutations in NBD1 alone decreased the open probability whereas mutations at NBD2 or simultaneously at both the NBDs (K464A/ K1250A) prolonged the frequency of bursts of activity. These data point out convincingly that the two NBD's cooperate to control channel gating. Login to comment

[hide] Effect of cystic fibrosis-associated mutations in ... J Biol Chem. 1996 Aug 30;271(35):21279-84. Cotten JF, Ostedgaard LS, Carson MR, Welsh MJ
Effect of cystic fibrosis-associated mutations in the fourth intracellular loop of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 1996 Aug 30;271(35):21279-84., [PMID:8702904]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) contains multiple membrane spanning sequences that form a Cl- channel pore and cytosolic domains that control the opening and closing of the channel. The fourth intracellular loop (ICL4), which connects the tenth and eleventh transmembrane spans, has a primary sequence that is highly conserved across species, is the site of a preserved sequence motif in the ABC transporter family, and contains a relatively large number of missense mutations associated with cystic fibrosis (CF). To investigate the role of ICL4 in CFTR function and to learn how CF mutations in this region disrupt function, we studied several CF-associated ICL4 mutants. We found that most ICL4 mutants disrupted the biosynthetic processing of CFTR, although not as severely as the most common DeltaF508 mutation. The mutations had no discernible effect on the channel's pore properties; but some altered gating behavior, the response to increasing concentrations of ATP, and stimulation in response to pyrophosphate. These effects on activity were similar to those observed with mutations in the nucleotide-binding domains, suggesting that ICL4 might help couple activity of the nucleotide-binding domains to gating of the Cl- channel pore. The data also explain how these mutations cause a loss of CFTR function and suggest that some patients with mutations in ICL4 may have a milder clinical phenotype because they retain partial activity of CFTR at the cell membrane.

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148 We found that two NBD1 mutants, K464A and G551S, had a normal or increased response to PPi (Fig. 7C). Login to comment
200 Data are mean Ϯ S.E. of (n) measurements for: wild-type (9), F1052V (3), R1066L (4), A1067T (4), G551S (6), K464A (4), G1349D (5), K1250 M at 5 mM PPi (5), wild-type at 5 mM PPi (16). Login to comment
147 We found that two NBD1 mutants, K464A and G551S, had a normal or increased response to PPi (Fig. 7C). Login to comment
199 Data are mean 6 S.E. of (n) measurements for: wild-type (9), F1052V (3), R1066L (4), A1067T (4), G551S (6), K464A (4), G1349D (5), K1250 M at 5 mM PPi (5), wild-type at 5 mM PPi (16). Login to comment

[hide] Glycerol reverses the misfolding phenotype of the ... J Biol Chem. 1996 Jan 12;271(2):635-8. Sato S, Ward CL, Krouse ME, Wine JJ, Kopito RR
Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation.
J Biol Chem. 1996 Jan 12;271(2):635-8., [PMID:8557666]

Abstract [show]
The common delta F508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) interferes with the biosynthetic folding of nascent CFTR polypeptides, leading to their retention and rapid degradation in an intracellular compartment proximal to the Golgi apparatus. Neither the pathway by which wild-type CFTR folds nor the mechanism by which the Phe508 deletion interferes with this process is well understood. We have investigated the effect of glycerol, a polyhydric alcohol known to stabilize protein conformation, on the folding of CFTR and delta F508 in vivo. Incubation of transient and stable delta F508 transfectants with 10% glycerol induced a significant accumulation of delta F508 protein bearing complex N-linked oligosaccharides, indicative of their transit to a compartment distal to the endoplasmic reticulum (ER). This accumulation was accompanied by an increase in mean whole cell cAMP activated chloride conductance, suggesting that the glycerol-rescued delta F508 polypeptides form functional plasma membrane CFTR channels. These effects were dose- and time-dependent and fully reversible. Glycerol treatment also stabilized immature (core-glycosylated) delta F508 and CFTR molecules that are normally degraded rapidly. These effects of glycerol were not due to a general disruption of ER quality control processes but appeared to correlate with the degree of temperature sensitivity of specific CFTR mutations. These data suggest a model in which glycerol serves to stabilize an otherwise unstable intermediate in CFTR biosynthesis, maintaining it in a conformation that is competent for folding and subsequent release from the ER quality control apparatus.

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95 Processing of mutants K464R and K464A was inefficient by comparison with wild type and was enhanced by incubation in the presence of 10% glycerol, even after accounting for the unequal label present in the immature precursor in the presence of glycerol (Fig. 3B). Login to comment
122 Interestingly, we observe a strong correlation between the temperature sensitivity of CFTR mutations like ⌬F508, K464R, and K464A (data not shown) and their ability to be remediated by glycerol. Login to comment
97 Processing of mutants K464R and K464A was inefficient by comparison with wild type and was enhanced by incubation in the presence of 10% glycerol, even after accounting for the unequal label present in the immature precursor in the presence of glycerol (Fig. 3B). Login to comment
124 Interestingly, we observe a strong correlation between the temperature sensitivity of CFTR mutations like DF508, K464R, and K464A (data not shown) and their ability to be remediated by glycerol. Login to comment

[hide] CFTR: the nucleotide binding folds regulate the ac... J Gen Physiol. 1996 Jan;107(1):103-19. Wilkinson DJ, Mansoura MK, Watson PY, Smit LS, Collins FS, Dawson DC
CFTR: the nucleotide binding folds regulate the accessibility and stability of the activated state.
J Gen Physiol. 1996 Jan;107(1):103-19., [PMID:8741733]

Abstract [show]
The functional roles of the two nucleotide binding folds, NBF1 and NBF2, in the activation of the cystic fibrosis transmembrane conductance regulator (CFTR) were investigated by measuring the rates of activation and deactivation of CFTR Cl- conductance in Xenopus oocytes. Activation of wild-type CFTR in response to application of forskolin and 3-isobutyl-1-methylxanthine (IBMX) was described by a single exponential. Deactivation after washout of the cocktail consisted of two phases: an initial slow phase, described by a latency, and an exponential decline. Rate analysis of CFTR variants bearing analogous mutations in NBF1 and NBF2 permitted us to characterize amino acid substitutions according to their effects on the accessibility and stability of the active state. Access to the active state was very sensitive to substitutions for the invariant glycine (G551) in NBF1, where mutations to alanine (A), serine (S), or aspartic acid (D) reduced the apparent on rate by more than tenfold. The analogous substitutions in NBF2 (G1349) also reduced the on rate, by twofold to 10-fold, but substantially destabilized the active state as well, as judged by increased deactivation rates. In the putative ATP-binding pocket of either NBF, substitution of alanine, glutamine (Q), or arginine (R) for the invariant lysine (K464 or K1250) reduced the on rate similarly, by two- to fourfold. In contrast, these analogous substitutions produced opposite effects on the deactivation rate. NBF1 mutations destabilized the active state, whereas the analogous substitutions in NBF2 stabilized the active state such that activation was prolonged compared with that seen with wild-type CFTR. Substitution of asparagine (N) for a highly conserved aspartic acid (D572) in the ATP-binding pocket of NBF1 dramatically slowed the on rate and destabilized the active state. In contrast, the analogous substitution in NBF2 (D1370N) did not appreciably affect the on rate and markedly stabilized the active state. These results are consistent with a hypothesis for CFTR activation that invokes the binding and hydrolysis of ATP at NBF1 as a crucial step in activation, while at NBF2, ATP binding enhances access to the active state, but the rate of ATP hydrolysis controls the duration of the active state. The relatively slow time courses for activation and deactivation suggest that slow processes modulate ATP-dependent gating.

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229 The calculated values of k,,~.for K464Q and K464A indicated that the substitutions to alanine or glutamine also increased the off rate under activating conditions, which contributed to the increase in Ka and compensated somewhat for the reduction in relaxation rate caused by the reduced on rate. Login to comment
281 + kott) (10-3 min-l kon kofr latency *k~m CFTR (mM) n (10-3min-]) mM-1) (10-3min 1) (10-3min-l) n (min) (10 3min i) n wt 0.65 • 0.08 26 664 • 51 118 • 9 558 • 45 76-+ 6 20 6.0 • 0.3 88 • 6 16 K464R 2.6 • 0.1": 4 153 + 20**+ 20 • 3*** 101 • 13''` 52 • 7*: 5 1.3 • 0.2*++ 174 • 14"** 7 K464Q 3.3 • 0.5"* 5 331 • 56*** 40 -+ 7* 199 • 34* 132 • 22*'` 5 1.9 • 0.3"I 142 -+ 19''` 5 K464A 4.6 • 0.7** 6 289 • 49* 30 • 5** 151 • 26*** 139 • 24*: 7 1.1 • 0.1"** 133 • 14"** 8 D572N 9.3 + 0.02*: 6 106 • 7*: 7-+0.5*: 37-+3*** 69 • 5+* 4 0.9 • 0.2*** 245 • 32*: 3 K1250R 0.17 • 0.07*: 5 239 •33*** 46 -+ 6"+* 231 • 32*: 8 • 1": 10 10.4 • 0.8"~ 100 • 7** 6 K1250Q 0.12 • 0.04*** 5 150 • 18''` 29 • 4* 146 -+ 18" 4 + 0.4"I 5 22.3 • 2.4*: 30 •5": 5 K1250A 0.07 + 0.02*: 10 218 • 18" 43 • 4*'` 215 • 18": 3 -+0.3*~* 5 15.6-+ 1.0"** 43 -+5** 5 D1370N 0.16 + 0.04*'` 7 449 - 79*: 87 • 15: 435 +76** 14 - 2*: 5 16.3-4-1.2"" 69-+ 6** 5 The symbols (*) and ('`) indicate significant differences from wild-type CFTR and the analogous mutant, respectively (P < 0.05). Login to comment
383 Similarly, the mutation K464A increased the interburst interval by fivefold, whereas rate analysis revealed a nearly fourfold slowing of the activation rate (k'o,1). Login to comment
231 The calculated values of k,,~.for K464Q and K464A indicated that the substitutions to alanine or glutamine also increased the off rate under activating conditions, which contributed to the increase in Ka and compensated somewhat for the reduction in relaxation rate caused by the reduced on rate. Login to comment
283 + kott) (10-3 min-l kon kofr latency *k~m CFTR (mM) n (10-3 min-]) mM-1) (10-3 min 1) (10-3min-l) n (min) (10 3min i) n wt 0.65 ߦ 0.08 26 664 ߦ 51 118 ߦ 9 558 ߦ 45 76 -+ 6 20 6.0 ߦ 0.3 88 ߦ 6 16 K464R 2.6 ߦ 0.1": 4 153 + 20**+ 20 ߦ 3*** 101 ߦ 13''` 52 ߦ 7*: 5 1.3 ߦ 0.2*++ 174 ߦ 14"** 7 K464Q 3.3 ߦ 0.5"* 5 331 ߦ 56*** 40 -+ 7* 199 ߦ 34* 132 ߦ 22*'` 5 1.9 ߦ 0.3"I 142 -+ 19''` 5 K464A 4.6 ߦ 0.7** 6 289 ߦ 49* 30 ߦ 5** 151 ߦ 26*** 139 ߦ 24*: 7 1.1 ߦ 0.1"** 133 ߦ 14"** 8 D572N 9.3 + 0.02*: 6 106 ߦ 7*: 7 -+0.5*: 37 -+3*** 69 ߦ 5+* 4 0.9 ߦ 0.2*** 245 ߦ 32*: 3 K1250R 0.17 ߦ 0.07*: 5 239 ߦ 33*** 46 -+ 6"+* 231 ߦ 32*: 8 ߦ 1": 10 10.4 ߦ 0.8"~ 100 ߦ 7** 6 K1250Q 0.12 ߦ 0.04*** 5 150 ߦ 18''` 29 ߦ 4* 146 -+ 18" 4 + 0.4"I 5 22.3 ߦ 2.4*: 30 ߦ 5": 5 K1250A 0.07 + 0.02*: 10 218 ߦ 18" 43 ߦ 4*'` 215 ߦ 18": 3 -+0.3*~* 5 15.6 -+ 1.0"** 43 -+5** 5 D1370N 0.16 + 0.04*'` 7 449 - 79*: 87 ߦ 15: 435 + 76** 14 - 2*: 5 16.3 -4-1.2"" 69 -+ 6** 5 The symbols (*) and ('`) indicate significant differences from wild-type CFTR and the analogous mutant, respectively (P < 0.05). Login to comment
385 Similarly, the mutation K464A increased the interburst interval by fivefold, whereas rate analysis revealed a nearly fourfold slowing of the activation rate (k'o,1). Login to comment

[hide] Conformational states of CFTR associated with chan... Cell. 1995 Jul 28;82(2):231-9. Gunderson KL, Kopito RR
Conformational states of CFTR associated with channel gating: the role ATP binding and hydrolysis.
Cell. 1995 Jul 28;82(2):231-9., [PMID:7543023]

Abstract [show]
CFTR is a member of the traffic ATPase superfamily and a Cl- ion channel that appears to require ATP hydrolysis for gating. Analysis of single CFTR Cl- channels reconstituted into planar lipid bilayers revealed the presence of two open conductance states that are connected to each other and to the closed state by an asymmetric cycle of gating events. We show here that the transition between the two open conductance states is directly coupled to ATP hydrolysis by one of the consensus nucleotide-binding folds, designated NBF2. Moreover, the transition between the closed state and one of the open states is linked to the binding of ATP. This analysis permits real-time visualization of conformational changes associated with a single cycle of ATP hydrolysis by a single protein molecule and suggests a model describing a role for ATP in CFTR gating.

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57 These NBF1 and NBF2 mutants, harboring the individual mutations K464A and K1250A, K1250G, K1250M, or K1250T, respectively, were expressed in HEK cells and reconstituted into planar lipid bilayers from which single-channel currents were recorded (Figures 3A and 3B). Login to comment
59 Compared with wild-type CFTR, the NBF1 mutant K464A exhibited slowed gating kinetics and reduced open probability (Po) in the presence of 1 mM MgATP (Figure 3A; see Figure 5). Login to comment
74 Effect of Mutation of the P Loop Lysine in NBF1 and NBF2 on CFTR Gating (A) Single-channel record of K464A in the absence (top) or presence (bottom)of 2 mM PP~. Login to comment
121 The simplest interpretation of these data is that ATP binding ConformationalStatesof CFTR 09 08 07 06 Oo 05 040.3 * 32 wildtype K464A K1250A G1247D/ D1370N 01249E 6~se' B 2500 20O0 5 1500 1OO0 500 o~ 0 w[Id{ype K464A K1250A C1247D/ Ol 370N G1249E Figure5. Login to comment
214 Polymerase Chain Reaction Megaprimer Mutagenesis The following site-directed mutants were constructed by using the megaprimer polymerase chain reaction (PCR)-based mutagenesis protocol (Landt et al., 1990; Sarkar and Sommer, 1990): K464A, K1250A, K1250G, K1250T, and GG1247, 1249DE. Login to comment
122 The simplest interpretation of these data is that ATP binding ConformationalStatesof CFTR 09 08 07 06 Oo 05 040.3 * 32 wildtype K464A K1250A G1247D/ D1370N 01249E 6~se' B 2500 20O0 5 1500 1OO0 500 o ~ 0 w[Id{ype K464A K1250A C1247D/ Ol 370N G1249E Figure5. Login to comment
215 Polymerase Chain Reaction Megaprimer Mutagenesis The following site-directed mutants were constructed by using the megaprimer polymerase chain reaction (PCR)-based mutagenesis protocol (Landt et al., 1990; Sarkar and Sommer, 1990): K464A, K1250A, K1250G, K1250T, and GG1247, 1249DE. Login to comment

[hide] Mechanism of dysfunction of two nucleotide binding... EMBO J. 1995 Mar 1;14(5):876-83. Sheppard DN, Ostedgaard LS, Winter MC, Welsh MJ
Mechanism of dysfunction of two nucleotide binding domain mutations in cystic fibrosis transmembrane conductance regulator that are associated with pancreatic sufficiency.
EMBO J. 1995 Mar 1;14(5):876-83., [PMID:7534226]

Abstract [show]
Variability in the severity of cystic fibrosis (CF) is in part due to specific mutations in the CF transmembrane conductance regulator (CFTR) gene. To understand better how mutations in CFTR disrupt Cl- channel function and to learn about the relationship between genotype and phenotype, we studied two CF mutants, A455E and P574H, that are associated with pancreatic sufficiency. A455E and P574H are located close to conserved ATP binding motifs in CFTR. Both mutants generated cAMP-stimulated apical membrane Cl- currents in heterologous epithelial cells, but current magnitudes were reduced compared with wild-type. Patch-clamp analysis revealed that both mutants had normal conductive properties and regulation by phosphorylation and nucleotides. These mutants had normal or increased Cl- channel activity: A455E had an open-state probability (Po) similar to wild-type, and P574H had an increased Po because bursts of activity were prolonged. However, both mutants produced less mature glycosylated protein, although levels were greater than observed with the delta F508 mutant. These changes in channel activity and processing provide a quantitative explanation for the reduced apical Cl- current. These data also dissociate structural requirements for channel function from features that determine processing. Finally, the results suggest that the residual function associated with these two mutants is sufficient to confer a milder clinical phenotype and infer approaches to developing treatments.

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177 It is interesting to compare the effect that different mutations in NBD1 had on al: P574H decreased a1, A455E did not change al, and K464A (a mutant of the Walker A lysine of NBDl; Carson and Welsh, 1995) appeared to increase the rate of channel closing. Login to comment

[hide] Exon 9 of the CFTR gene: splice site haplotypes an... Hum Genet. 1994 Jan;93(1):67-73. Dork T, Fislage R, Neumann T, Wulf B, Tummler B
Exon 9 of the CFTR gene: splice site haplotypes and cystic fibrosis mutations.
Hum Genet. 1994 Jan;93(1):67-73., [PMID:7505767]

Abstract [show]
The alternatively spliced exon 9 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene codes for the initial part of the amino-terminal nucleotide-binding fold of CFTR. A unique feature of the acceptor splice site preceding this exon is a variable length polymorphism within the polypyrimidine tract influencing the extent of exon 9 skipping in CFTR mRNA. We investigated this repeat for its relationship to CFTR mutations and intragenic markers on 200 chromosomes from German patients with cystic fibrosis (CF). Four frequent length variations were strongly associated with the four predominant haplotypes previously defined by intragenic marker dimorphisms. One of these alleles displayed absolute linkage disequilibrium to the major CF mutation delta F508. Other frequent CFTR mutations were linked to one particular splice site haplotype indicating that differential exon 9 skipping contributes little to the clinical heterogeneity among CF patients with an identical mutation. We also identified a novel missense mutation (V456F) and a novel nonsense mutation (Q414X) within the coding region of exon 9. The missense mutation V456F adjacent to Walker motif A was present in a pancreas-sufficient CF patient. In contrast, the pancreas-insufficient Q414X/delta F508 compound heterozygote suffered from a severe form of the disease, indicating that alternative splicing of exon 9 does not overcome the deleterious effect of a stop codon with this exon.

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115 Early evidence for a crucial role of exon 9 sequences came from the findings that mutations 71 within or adjacent to the Walker motif A produce a CF phenotype in vivo (A455E, G458V; Kerem et al. 1990; Cuppens et al. 1990) and in vitro (K464A; Anderson and Welsh 1992). Login to comment

[hide] Functional roles of the nucleotide-binding folds i... Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9963-7. Smit LS, Wilkinson DJ, Mansoura MK, Collins FS, Dawson DC
Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator.
Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9963-7., [PMID:7694298]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the traffic ATPase superfamily, possesses two putative nucleotide-binding folds (NBFs). The NBFs are sufficiently similar that sequence alignment of highly conserved regions can be used to identify analogous residues in the two domains. To determine whether this structural homology is paralleled in function, we compared the activation of chloride conductance by forskolin and 3-isobutyl-1-methylxanthine in Xenopus oocytes expressing CFTRs bearing mutations in NBF1 or NBF2. Mutation of a conserved glycine in the putative linker domain in either NBF produced virtually identical changes in the sensitivity of chloride conductance to activating conditions, and mutation of this site in both NBFs produced additive effects, suggesting that in the two NBFs this region plays a similar and critical role in the activation process. In contrast, amino acid substitutions in the Walker A and B motifs, thought to form an integral part of the nucleotide-binding pockets, produced strikingly different effects in NBF1 and NBF2. Substitutions for the conserved lysine (Walker A) or aspartate (Walker B) in NBF1 resulted in a marked decrease in sensitivity to activation, whereas the same changes in NBF2 produced an increase in sensitivity. These results are consistent with a model for the activation of CFTR in which both NBF1 and NBF2 are required for normal function but in which either the nature or the exact consequences of nucleotide binding differ for the two domains.

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89 Alanine and arginine substitutions at lysine-464 and -1250 were associated with sensitivities similar to those observed with the glutamine substitutions (K464A or K464R, Kil2 = 0.8 mM; K1250A or K1250R, K,12 < 0.02 mM). Login to comment

[hide] Role of CFTR's PDZ1-binding domain, NBF1 and Cl(-)... Biochim Biophys Acta. 2001 Nov 1;1515(1):64-71. Boucherot A, Schreiber R, Kunzelmann K
Role of CFTR's PDZ1-binding domain, NBF1 and Cl(-) conductance in inhibition of epithelial Na(+) channels in Xenopus oocytes.
Biochim Biophys Acta. 2001 Nov 1;1515(1):64-71., [PMID:11597353]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) inhibits epithelial Na(+) channels (ENaC). Evidence has accumulated that both Cl(-) transport through CFTR Cl(-) channels and the first nucleotide binding domain (NBF1) of CFTR are crucial for inhibition of ENaC. A PDZ binding domain (PDZ-BD) at the C-terminal end links CFTR to scaffolding and cytoskeletal proteins, which have been suggested to play an important role in activation of CFTR and eventually inhibition of ENaC. We eliminated the PDZ-BD of CFTR and coexpressed Na(+)/H(+)-exchange regulator factors together with CFTR and ENaC. The results do not support a role of PDZ-BD in inhibition of ENaC by CFTR. However, inhibition of ENaC was closely linked to Cl(-) currents generated by CFTR and was observed in the presence of Cl(-), I(-) or Br(-) but not gluconate. Therefore, functional NBF1 and Cl(-) transport are required for inhibition of ENaC in Xenopus oocytes, while the PDZ-BD is not essential.

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38 Using similar PCR techniques, the NBF1 mutants of human CFTR vF508, G551D, S466L, K464A, D572N, KH483/484AA, R487Q, R516A, KR598/600GA, KK611/612AA and K615A were in vitro synthesized (Quickchange, Stratagene). Login to comment
171 We therefore introduced mutations into NBF1 sites which are essential for binding/hydrolysis of ATP and GTP and which have homology to GTP binding proteins such as Walker A (loop L1) (K464A, S466L), switch I motif (KH483/484AA, R487A), switch II motif (loop L4, G551D) and Walker B (D572N) [23]. Login to comment

[hide] An intrinsic adenylate kinase activity regulates g... Cell. 2003 Dec 26;115(7):837-50. Randak C, Welsh MJ
An intrinsic adenylate kinase activity regulates gating of the ABC transporter CFTR.
Cell. 2003 Dec 26;115(7):837-50., [PMID:14697202]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP binding cassette (ABC) transporter family. Like other ABC transporters, it can hydrolyze ATP. Yet while ATP hydrolysis influences channel gating, it has long seemed puzzling that CFTR would require this reaction because anions flow passively through CFTR. Moreover, no other ion channel is known to require the large energy of ATP hydrolysis to gate. We found that CFTR also has adenylate kinase activity (ATP + AMP <=> ADP + ADP) that regulates gating. When functioning as an adenylate kinase, CFTR showed positive cooperativity for ATP suggesting its two nucleotide binding domains may dimerize. Thus, channel activity could be regulated by two different enzymatic reactions, ATPase and adenylate kinase, that share a common ATP binding site in the second nucleotide binding domain. At physiologic nucleotide concentrations, adenylate kinase activity, rather than ATPase activity may control gating, and therefore involve little energy consumption.

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263 The Walker A mutations (K464A in NBD1 and K1250A in stimulation (Berger et al., 2002). Login to comment
269 Thus, these data begin to discriminate between the ATPase and adenylate kinase effects whereas K464A enhanced Ap5A inhibition (Figures 7E and 7F). Login to comment
288 Data are from 5 (wild-type, K464A, D572N), 9 (K1250A), 10 (D1370N), and 3 (N1303K) membrane patches. Asterisks indicate p b0d; 0.05 compared to wild-type by ANOVA followed by Dunnett`s multiple comparison test. Login to comment

[hide] Conformational changes in the catalytically inacti... J Gen Physiol. 2013 Jul;142(1):61-73. doi: 10.1085/jgp.201210954. Epub 2013 Jun 10. Csanady L, Mihalyi C, Szollosi A, Torocsik B, Vergani P
Conformational changes in the catalytically inactive nucleotide-binding site of CFTR.
J Gen Physiol. 2013 Jul;142(1):61-73. doi: 10.1085/jgp.201210954. Epub 2013 Jun 10., [PMID:23752332]

Abstract [show]
A central step in the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the association of its two cytosolic nucleotide-binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric adenosine triphosphate (ATP)-binding cassette protein in which the two interfacial-binding sites (composite sites 1 and 2) are functionally different. During gating, the canonical, catalytically active nucleotide-binding site (site 2) cycles between dimerized prehydrolytic (state O1), dimerized post-hydrolytic (state O2), and dissociated (state C) forms in a preferential C-->O1-->O2-->C sequence. In contrast, the catalytically inactive nucleotide-binding site (site 1) is believed to remain associated, ATP-bound, for several gating cycles. Here, we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1. Previous work showed that channel closure is slowed, both under hydrolytic and nonhydrolytic conditions, by occupancy of site 1 by N(6)-(2-phenylethyl)-ATP (P-ATP) as well as by the site-1 mutation H1348A (NBD2 signature sequence). Here, we found that P-ATP prolongs wild-type (WT) CFTR burst durations by selectively slowing (>2x) transition O1-->O2 and decreases the nonhydrolytic closing rate (transition O1-->C) of CFTR mutants K1250A ( approximately 4x) and E1371S ( approximately 3x). Mutation H1348A also slowed ( approximately 3x) the O1-->O2 transition in the WT background and decreased the nonhydrolytic closing rate of both K1250A ( approximately 3x) and E1371S ( approximately 3x) background mutants. Neither P-ATP nor the H1348A mutation affected the 1:1 stoichiometry between ATP occlusion and channel burst events characteristic to WT CFTR gating in ATP. The marked effect that different structural perturbations at site 1 have on both steps O1-->C and O1-->O2 suggests that the overall conformational changes that CFTR undergoes upon opening and coincident with hydrolysis at the active site 2 include significant structural rearrangement at site 1.

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29 For instance, the K464A mutation, which-by removing the side chain of the conserved NBD1 Walker A lysine-perturbs the NBD1 side of site 1, dramatically alters the mechanism of gating (Csan&#e1;dy et al., 2010). Login to comment
35 M A T E R I A L S A N D M E T H O D S Molecular biology Human WT CFTR and CFTR segment 433-1480 in the pGEMHE plasmid (Chan et al., 2000) served as templates for mutants H1348A, K1250A, E1371S, K1250A/H1348A, E1371S/H1348A, E1371S/K464A, and 433-1480(K1250A), which were created using the QuikChange kit (Agilent Technologies). Login to comment
66 Fig. S2 shows the effect of the K464A mutation on nonhydrolytic closing rate measured in the E1371S background. Login to comment
219 Interestingly, the K464A mutation, which perturbs site 1 by removing the conserved Walker A lysine, was also shown to affect the energetics of both of the C1࢒O1 and O1࢐O2 gating steps (Csan&#e1;dy et al., 2010), although in a different way: in this mutant, rate k1 decreased approximately fourfold, whereas the rate of nonhydrolytic closure, in a K1250A mutant background, increased by &#e07a;10-fold (this is also replicated in the E1371S background; Fig. S2). Login to comment
220 Thus, although the distortion of the energetic profile by the K464A mutation differs from that seen for the two perturbations studied here, the K464A result is again consistent with movements in site 1 occurring in both of the above gating steps. Login to comment

[hide] ATP and AMP mutually influence their interaction w... J Biol Chem. 2013 Sep 20;288(38):27692-701. doi: 10.1074/jbc.M113.479675. Epub 2013 Aug 6. Randak CO, Dong Q, Ver Heul AR, Elcock AH, Welsh MJ
ATP and AMP mutually influence their interaction with the ATP-binding cassette (ABC) adenylate kinase cystic fibrosis transmembrane conductance regulator (CFTR) at separate binding sites.
J Biol Chem. 2013 Sep 20;288(38):27692-701. doi: 10.1074/jbc.M113.479675. Epub 2013 Aug 6., [PMID:23921386]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP &lrarr2; 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5'-triphosphate (8-N3-ATP) and 8-azidoadenosine 5'-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P(1),P(5)-di(adenosine-5') pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.

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275 Error bars, S.E. Nucleotide Interactions with the ABC Adenylate Kinase CFTR 27698 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288ߦNUMBER 38ߦSEPTEMBER 20, 2013 at SEMMELWEIS UNIV OF MEDICINE on December , D1370N, abolished Ap5A inhibition of current, whereas the homologous mutations in ATP-binding site 1, K464A and D572N, did not. Login to comment
303 However, the homologous mutations in ATP-binding site 1 (K464A and D572N) did not (19). Login to comment