ABCMdb

ABCC7 p.Lys1250Met

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 (95%), 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] 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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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

[hide] Down-regulation of volume-sensitive Cl- channels b... Pflugers Arch. 2002 Nov;445(2):177-86. Epub 2002 Sep 7. Ando-Akatsuka Y, Abdullaev IF, Lee EL, Okada Y, Sabirov RZ
Down-regulation of volume-sensitive Cl- channels by CFTR is mediated by the second nucleotide-binding domain.
Pflugers Arch. 2002 Nov;445(2):177-86. Epub 2002 Sep 7., [PMID:12457238]

Abstract [show]
Transient expression of wild-type human cystic fibrosis transmembrane conductance regulator (CFTR) in HEK293T cells resulted in a profound decrease in the amplitude of volume-sensitive outwardly rectifying Cl- channel (VSOR) current without changing the single-channel amplitude. This effect was not mimicked by expression of the DeltaF508 mutant of CFTR, which did not reach the plasma membrane. The VSOR regulation by CFTR was not affected by G551D mutation at first nucleotide-binding domain (NBD1), which is known to impair CFTR interaction with the outwardly rectifying chloride channel, ORCC, epithelial amiloride-sensitive Na-channel, ENaC, and renal potassium channel, ROMK2. The CFTR-VSOR interaction was insensitive to the deletion mutation, DeltaTRL, which is known to impair CFTR-PDZ domain binding. In contrast, the G1349D mutant, which impairs ATP binding at NBD2, effectively abolished the down-regulatory effect of CFTR. Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Thus, we conclude that an ATP-hydrolysable conformation of NBD2 is essential for the regulation of the VSOR by the CFTR protein, and that VSOR is a first channel regulated by CFTR through its NBD2.

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6 Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Login to comment
123 To check whether ATP hydrolysis at NBD2 is required for CFTR`s regulatory function, we generated two other NBD2-mutants of CFTR, K1250M and D1370N. Login to comment
125 The plasmalemmal distribution of K1250M and D1370N CFTR was confirmed by immunofluorescence microscopy (Fig. 6A) and immunoblotting (Fig. 6B). Login to comment
131 Relative integrated optical densities of the mature bands are shown as percentages of the respective b-actin bands (taken as 100%) In whole-cell patch-clamp experiments, cells expressing the K1250M or D1370N mutant exhibited VSOR currents (Fig. 7A) as large as those observed in mock-transfected cells (Fig. 3A). Login to comment
137 Since VSOR-non-regulating NBD2 mutants (G1349D, K1250M and D1370N) were expressed to approximately the same level as VSOR-regulating WT and G551D CFTR (as seen from immunostaining and Western blotting data), we may exclude the possibility that the down-regulation is simply a side-effect of overexpression of a foreign protein. Login to comment
155 This was, in fact, confirmed by a yeast two- Fig. 7A, B Effects of expression of Walker A or Walker B mutants on VSOR current densities in HEK293T cells. A Time course of VSOR current activation by hypotonic stimulation of cells transfected with the K1250M (top) or D1370N (bottom) mutant, taken during application of alternating pulses from 0 to €40 mV every 15 s. B VSOR current densities from mock-transfected, K1250M mutant-transfected and D1370N mutant-transfected cells, recorded at +40 mV after reaching a steady-state level. Login to comment
175 In our study, the K1250M and D1370N mutations effectively abolished the down-regulatory effect of CFTR on VSOR currents. 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
46 These included (i) mouse mammary epithelial cells (C127 cells) expressing wild-type human CFTR, the CFTR variant ⌬R-S660A (13) or the CF mutant G1349D (14), (ii) Fischer rat thyroid epithelial cells expressing the CF mutant G551D (15), and (iii) NIH-3T3 cells expressing the CFTR construct K1250M (16). 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
232 By contrast, the pHi sensitivity of K1250M-CFTR differed strikingly from that of wild-type CFTR. Login to comment
233 At pHi 6.3, the Po of K1250M-CFTR was reduced because MBD was decreased 0.7-fold, whereas IBI was unchanged (Figs. 7, G and H, and 8). Login to comment
234 At pHi 8.3, the Po of K1250M-CFTR was increased, albeit slightly, as a result of a small increase in MBD and no change in IBI (Figs. 7H and 8). Login to comment
235 Fig. 7H also reveals that the Po of K1250M-CFTR was similar to that of wild-type CFTR at pHi 8.3, but greatly diminished at pHi 7.3 and 6.3. Login to comment
236 Thus, the pHi sensitivity of K1250M-CFTR is the converse of that of wild-type 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
284 Burst analysis of D572N- and K1250M-CFTR. Login to comment
285 A and B, MBD and IBI of D572N- and K1250M-CFTR at different pHi values; wild-type CFTR data are shown for comparison. Login to comment
286 Data are means Ϯ S.E. (D572N- and K1250M-CFTR, n ϭ 3; wild-type-CFTR, n Ն 6). Login to comment
312 By contrast, Hϩ ions are either without effect (D1370N-CFTR) or inhibit (K1250M-CFTR) the gating behavior of site-directed mutations in ATP-binding site 2. Login to comment
343 The pHi Sensitivity of CFTR Reveals Cross-talk between ATP Binding Sites 1 and 2-An interesting aspect of our data is the pHi sensitivity of K1250M-CFTR, which is the reverse of that of wild-type CFTR. Login to comment
345 If K1250M-CFTR severely disrupts the function of site 2 (16), these hidden minor pHi effects might originate from site 1. Login to comment

[hide] Regulation of Cl-/ HCO3- exchange by cystic fibros... J Biol Chem. 1999 Feb 5;274(6):3414-21. Lee MG, Wigley WC, Zeng W, Noel LE, Marino CR, Thomas PJ, Muallem S
Regulation of Cl-/ HCO3- exchange by cystic fibrosis transmembrane conductance regulator expressed in NIH 3T3 and HEK 293 cells.
J Biol Chem. 1999 Feb 5;274(6):3414-21., 1999-02-05 [PMID:9920885]

Abstract [show]
A central function of cystic fibrosis transmembrane conductance regulator (CFTR)-expressing tissues is the secretion of fluid containing 100-140 mM HCO3-. High levels of HCO3- maintain secreted proteins such as mucins (all tissues) and digestive enzymes (pancreas) in a soluble and/or inactive state. HCO3- secretion is impaired in CF in all CFTR-expressing, HCO3--secreting tissues examined. The mechanism responsible for this critical problem in CF is unknown. Since a major component of HCO3- secretion in CFTR-expressing cells is mediated by the action of a Cl-/HCO3- exchanger (AE), in the present work we examined the regulation of AE activity by CFTR. In NIH 3T3 cells stably transfected with wild type CFTR and in HEK 293 cells expressing WT and several mutant CFTR, activation of CFTR by cAMP stimulated AE activity. Pharmacological and mutagenesis studies indicated that expression of CFTR in the plasma membrane, but not the Cl- conductive function of CFTR was required for activation of AE. Furthermore, mutations in NBD2 altered regulation of AE activity by CFTR independent of their effect on Cl- channel activity. At very high expression levels CFTR modified the sensitivity of AE to 4,4'-diisothiocyanatostilbene-2, 2'-disulfonate. The novel finding of regulation of Cl-/HCO3- exchange by CFTR reported here may have important physiological implications and explain, at least in part, the impaired HCO3- secretion in CF.

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No. Sentence Comment
52 The mutagenesis primers were as follows: P205S primer, 5Ј-CGT GTG GAT CGC TTC TTT GCA AGT GGC-3Ј; W846term, 5Ј-GAG CAT ACC AGC AGT GAC TAC ATA GAA CAC ATA CCT TCG ATA TAT TAC-3Ј; G1247D/G1249E, 5Ј-GTG GGC CTC TTG GGA AGA ACT GAT TCA GAG AAG AGT ACT TTG TTA TCA GC-3Ј; K1250M, 5Ј-CTT GGG AAG AAC TGG ATC AGG GAT GAG TAC TTT GTT ATC AGC-3Ј; D1370N, 5Ј-GTA AGG CGA AGA TCT TGC TGC TTA ATG AAC CCA GTG CTC ATT TGG ATC-3Ј. Login to comment
163 Fig. 6 (i-k) shows the plasma membrane localization of K1250M CFTR, D1370N CFTR, and the double mutant G1247D/ G1249E CFTR, respectively. Login to comment
240 The K1250M CFTR mutant had increased channel activity (32), was expressed in the plasma membrane (Fig. 6i) and activated AE similar to WT CFTR (Fig. 12b). Login to comment
265 The K1250M mutant was at least as effective as WT CFTR in stimulating AE activity (b). 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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471 Although this would seem to rulestill able to open CFTR channels with a mutation, K1250M, in the NBD2 Walker A motif that was expected to impair out the postulated close structural relationship between G proteins and CFTR`s NBDs, the assignment of their rolesATP hydrolysis there (3). 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] Cystic fibrosis: a multiple exocrinopathy caused b... Am J Med. 1998 Jun;104(6):576-90. Schwiebert EM, Benos DJ, Fuller CM
Cystic fibrosis: a multiple exocrinopathy caused by dysfunctions in a multifunctional transport protein.
Am J Med. 1998 Jun;104(6):576-90., [PMID:9674722]

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224 In NBD2, a few key mutations have been found that include missense mutations (G1349D, D1370N, K1250M, K1250Q, G1244E, S1255P) and several nonsense mutations (W1282X, S1255X, W1316X). 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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178 We found that F- stimulates CFTR-K1250M channels to a greater extent than wild-type CFTR (Fig. 8). Login to comment
221 More strikingly, PPi stimulated less current in K1250M-CFTR than in wild-type CFTR (14), whereas F- stimulated more current in K1250M-CFTR than in wild-type CFTR. Therefore, there are distinct differences between the effects of F- and PPi. Login to comment
230 Effect of increasing F- concentration on current in wild-type CFTR, CFTR-K464A, and CFTR-K1250M. Login to comment
171 We found that F2 stimulates CFTR-K1250M channels to a greater extent than wild-type CFTR (Fig. 8). Login to comment
213 More strikingly, PPi stimulated less current in K1250M-CFTR than in wild-type CFTR (14), whereas F2 stimulated more current in K1250M-CFTR than in wild-type CFTR. Therefore, there are distinct differences between the effects of F2 and PPi. Login to comment
222 Effect of increasing F2 concentration on current in wild-type CFTR, CFTR-K464A, and CFTR-K1250M. Login to comment

[hide] Function of the R domain in the cystic fibrosis tr... J Biol Chem. 1997 Oct 31;272(44):28133-41. Ma J, Zhao J, Drumm ML, Xie J, Davis PB
Function of the R domain in the cystic fibrosis transmembrane conductance regulator chloride channel.
J Biol Chem. 1997 Oct 31;272(44):28133-41., [PMID:9346969]

Abstract [show]
For a cystic fibrosis transmembrane conductance regulator (CFTR) channel to enter its open state, serine residues in the R domain must be phosphorylated by cAMP-dependent protein kinase, and intracellular ATP must bind to the nucleotide-binding folds and subsequently be hydrolyzed. CFTR with its R domain partially removed, DeltaR(708-835)-CFTR, forms a chloride channel that opens independently of protein kinase A phosphorylation, with open probability approximately one-third that of the wild type CFTR channel. Deletion of this portion of the R domain from CFTR alters the response of the channel to 5'-adenylylimidodiphosphate, pyrophosphate, and vanadate, compounds that prolong burst duration of the wild type CFTR channel but fail to do so in the DeltaR-CFTR. In addition, the addition of exogenous unphosphorylated R domain protein, which blocks the wild type CFTR channel, has no effect on the DeltaR-CFTR channel. However, when the exogenous R domain is phosphorylated, significant stimulation of the DeltaR-CFTR channel results; Po increases from 0.10 to 0.22. These data are consistent with a model for CFTR function in which the R domain in the unphosphorylated state interacts with the first nucleotide binding fold to inhibit either binding or hydrolysis of ATP or transduction of the effect to open the pore, but when the R domain is phosphorylated, it undergoes conformational change and interacts at a separate site in the first nucleotide binding fold to stimulate either binding or hydrolysis of ATP or transduction of the effect to open the pore.

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No. Sentence Comment
258 Point mutations within the conserved Walker A motif of NBF1 decreased the opening rate of the CFTR channel, while the corresponding mutations in NBF2 (K1250A, K1250M) prolong the open lifetime of CFTR (14, 15). Login to comment
259 The functional effects of K1250A and K1250M on the CFTR channel are similar to the effects of AMP-PNP and PPi (19, 24), suggesting that a decrease in the ATP hydrolysis rate at NBF2 leads to prolonged opening of the CFTR channel. Login to comment
260 The functional effects of K1250A and K1250M on the CFTR channel are similar to the effects of AMP-PNP and PPi (19, 24), suggesting that a decrease in the ATP hydrolysis rate at NBF2 leads to prolonged opening of the CFTR channel. 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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No. Sentence Comment
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

[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
149 In contrast, mutation of two analogous residues in NBD2, K1250M TABLE I Effect of ICL4 mutations on anion selectivity Channels were activated by PKA and 1 mM ATP. Login to comment

[hide] Epitope tagging permits cell surface detection of ... Am J Physiol. 1995 Dec;269(6 Pt 1):C1565-76. Howard M, DuVall MD, Devor DC, Dong JY, Henze K, Frizzell RA
Epitope tagging permits cell surface detection of functional CFTR.
Am J Physiol. 1995 Dec;269(6 Pt 1):C1565-76., [PMID:8572187]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated Cl channel responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-induced Cl secretion across the apical membranes of epithelial cells. To optimize its detection for membrane localization studies, we tagged CFTR with epitope sequences at the carboxy terminus or in the fourth external loop. When epitopes were added to the fourth external loop, the N-linked glycosylation sites in that loop were either preserved or they were mutated to produce a deglycosylated CFTR (dgCFTR). Tagged CFTRs were expressed in HeLa cells, and their cAMP-sensitive Cl permeability was assayed using the halide-sensitive fluorophore SPQ. CFTRs containing the M2 epitope showed halide permeability responses to cAMP, whereas cells expressing CFTR with the hemagglutinin (HA) tag showed little or no cAMP response. Xenopus oocytes expressing dgCFTR, with or without the M2 epitope, showed Cl conductance responses that were 20% of the wild-type response, whereas M2-tagged constructs retaining the glycosylation sites responded like wild-type CFTR. External M2-tagged CFTR was detected in the surface membrane of nonpermeabilized cells. The surface expression of the mutant M2-tagged CFTRs correlated with processing of these mutants (Gregory et al. Mol. Cell. Biol. 11:3886-3893, 1991). M2-901/CFTR is a useful reporter for the trafficking of wild-type and mutant CFTRs to the cell surface.

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251 M2-901 CFIR 5 0 -1 mV C constructed two dgCFTR mutants that are not associated with disease, K1250M and D1370N, also bearing This is a technical study that identifies sites appropri- the M2 epitope at position 901. Login to comment
255 Cell surface expression of M2-901/CFTR has been observed in a expression of M2-901-labeled G551D, G1349D, K1250M, and D1370N dgCFTRs correlates with carbohydrate variety of cell types including HEp-2, BSC40, and addition (Fig &A-II). Login to comment
265 A: G551D; B: G1349D; C: K1250M; D: D1370N; E: AF508; F: N1303K. 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
68 The mean conductance of K1250M channels was 8.9 __. 0.13 (n = 4) pS (Figures 3C and 3D), which is not significantly different from the conductance of the wild-type O1 state. Our data show that the O~--*O= transition is highly asymmetric in wild-type channels, consistent with the large free energy change associated with ATP hydrolysis. Login to comment
70 c KI250G ' C K1250M - :~:-~ ~ . Login to comment
76 (B)Single-channelrecordsof K1250A,K1250G, K1250M, and K1250T filtered at 50 Hz under standard cis bath conditions. Login to comment
241 Acknowledgments Correspondence should be addressed to R. R. K. We thank M. Welsh for kindly providing the K1250M and D1370N mutants, P. Quinton for his insightful discussions and critical reading of the manuscript, C. Login to comment
242 Acknowledgments Correspondence should be addressed to R. R. K. We thank M. Welsh for kindly providing the K1250M and D1370N mutants, P. Quinton for his insightful discussions and critical reading of the manuscript, C. Login to comment

[hide] The CFTR chloride channel of mammalian heart. Annu Rev Physiol. 1995;57:387-416. Gadsby DC, Nagel G, Hwang TC
The CFTR chloride channel of mammalian heart.
Annu Rev Physiol. 1995;57:387-416., [PMID:7539989]

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110 The conclusion is that ATP hydrolysis, most likely at NBD1 (because ATP regulates gating of an NBD2 mutant, K1250M), is required to open CFfR channels (2). Login to comment

[hide] Inhibition of the cystic fibrosis transmembrane co... Ann N Y Acad Sci. 1993 Dec 20;707:275-84. Sheppard DN, Welsh MJ
Inhibition of the cystic fibrosis transmembrane conductance regulator by ATP-sensitive K+ channel regulators.
Ann N Y Acad Sci. 1993 Dec 20;707:275-84., [PMID:9137558]

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68 We studied CFTR-containing mutations that affect each of the three types of domains of CFTR: K3 35E, which contains a mutation in the sixth transmembrane segment; K1250M, which contains a mutation in the second NBD; and CFTRAR, where part of the R domain has been deleted (amino acids 708-835). 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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129 Of particular interest is the observation (28) that mutation of the Walker A lysine in NBF2 to methionine (K1250M) produced a 4-fold reduction in the apparent Kil2 for the activation of CFTR by ATP but also completely eliminated inhibition by 1 mM ADP. Login to comment

[hide] Effect of ATP-sensitive K+ channel regulators on c... J Gen Physiol. 1992 Oct;100(4):573-91. Sheppard DN, Welsh MJ
Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents.
J Gen Physiol. 1992 Oct;100(4):573-91., [PMID:1281220]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by cAMP-dependent phosphorylation and by intracellular ATP. Intracellular ATP also regulates a class of K+ channels that have a distinct pharmacology: they are inhibited by sulfonylureas and activated by a novel class of drugs called K+ channel openers. In search of modulators of CFTR Cl- channels, we examined the effect of sulfonylureas and K+ channel openers on CFTR Cl- currents in cells expressing recombinant CFTR. The sulfonylureas, tolbutamide and glibenclamide, inhibited whole-cell CFTR Cl- currents at half-maximal concentrations of approximately 150 and 20 microM, respectively. Inhibition by both agents showed little voltage dependence and developed slowly; > 90% inhibition occurred 3 min after adding 1 mM tolbutamide or 100 microM glibenclamide. The effect of tolbutamide was reversible, while that of glibenclamide was not. In contrast to their activating effect on K+ channels, the K+ channel openers, diazoxide, BRL 38227, and minoxidil sulfate inhibited CFTR Cl- currents. Half-maximal inhibition was observed at approximately 250 microM diazoxide, 50 microM BRL 38227, and 40 microM minoxidil sulfate. The rank order of potency for inhibition of CFTR Cl- currents was: glibenclamide < BRL 38227 approximately equal to minoxidil sulfate > tolbutamide > diazoxide. Site-directed mutations of CFTR in the first membrane-spanning domain and second nucleotide-binding domain did not affect glibenclamide inhibition of CFTR Cl- currents. However, when part of the R domain was deleted, glibenclamide inhibition showed significant voltage dependence. These agents, especially glibenclamide, which was the most potent, may be of value in identifying CFTR Cl- channels. They or related analogues might also prove to be of value in treating diseases such as diarrhea, which may involve increased activity of the CFTR Cl- channel.

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46 MATERIALS AND METHODS For this study we used NIH 3T3 fibroblasts that had been stably infected with a retrovirus expressing either wild-type human CFTR (Gregory, Cheng, Rich, Marshall, Paul, Hehir, Ostedgaard, Klinger, Welsh, and Smith, 1990) or a CFTR mutant in NBD2 (CFIR-KI250M, in which lysine 1250 was changed to methionine) (Anderson et al., 1991a). Login to comment
161 We therefore examined the effect of glibenclamide, the most potent inhibitor we had identified, on CI- currents generated by several CFFR mutants. We studied 100 • 1992 CFTR containing mutations that affect each of the three types of domains of CFTR: CFFP~R where part of the R domain has been deleted (amino acids 708-835); CVFR-K335E, which contains a mutation in the sixth putative membrane-spanning sequence; and CFTR-K1250M, which contains a mutation in the second NBD. Login to comment
173 Comparison with ATP-dependent transporters suggest that mutations in the second NBD, such as CFTR-K1250M, are likely to abolish or impair the function of SHEPP~LRDANDWELSHK-ATP Channel Regulators Inhibit CFTR NBD2 (Anderson et al., 1991a). Login to comment
174 Nevertheless, CFFR-K1250M produces CI- channels that have cAMP-dependent regulation and biophysical properties that are similar to wild-type CFFR (see Anderson et al., 1991a, and Fig. 7, G and I). Login to comment
175 Inhibition of CFTR-K1250M CI- currents by glibenclamide was similar to that observed with wild-type CYI'R (Fig. 7, H and I). Login to comment
176 +SO mV 0 m V ~ -go mV CFTFI~ A~ ......~-1 baseline D CFTR-K335E I ii..i.Iii cAMP .... 13, CFTR-K1250M 1cAMP baseline + gllbenclamlde 250 pA L 50 ms C 1500 1000 5OO -500 o1~0 -100 -75 -50 -25 0 V (mV) ---o-- baseline ---4-- gllbenclamlde E) I cAMP + gllbenclamlde / tp----e F 1000 750 5OO -100 -75 -50 -25 0 V (mV) H ) 1000 PAL__ 50 ms I i cAMP + gllbenclamlde --o-- cAMP --e-- gllbanclamlde / _~_ _ 4OOO 3OOO ~q]O0 I000 0 I000 2OOO 30OO mOO -100 -75 -50 -25 0 V (mV) 25 50 25 50 25 50 FIGURE 7. Login to comment
178 Traces are from C127 cells stably expressing CFTP-~R (A-C), HeLa cells transiently expressing CFFR-K335E (D-F), and NIH 3T3 cells stably expressing CFIR-K1250M (G-I). Login to comment
180 CFFRAR currents were recorded in the absence of cAMP, but for CFFR-K335E and CFTR-K1250M currents cAMP agonists were present. Login to comment
185 Data for steady-state current values measured at +50 and -90 mV are shown for wild-type CF-FR (A), CFTRAR (B), CFFR-K335E (C), and CFTR-K1250M (D). Login to comment
186 The dose-response curves for wild-type CFFR, CFTR-K335E, and CFTR-K1250M were similar. Login to comment
187 Although the effect was not marked, in each case inhibition was slightly more potent at -90 mV than at +50 inV. A 100 8O O 60 c g 20 B 100 8O o 6o eo o m 20 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992 C CFTR 100 ' --o-- 50mV 80 e 6o C g,m 2O 0 20 40 60 80 1C10 0 gllbenclamlde (,aM) D CFTRAR 100 80 2 60 c i 2O 1 ~ ~ 866o o glil~nclamlde (gM) CFTR-K335E 0 20 40 ~ 80 100 glibenclamlda (,ttM) CFTR-K1250M "il o 2i~ ,o 6i) 8o lOO glibenclamlde (i.tM) FIGURE 8. Login to comment
191 (A) Wild-type CFTR; (B) CFTR~R; (C) CFTR-K335E; (D) CFFR-K1250M. Login to comment
196 Half-maximal inhibition by glibenclamide was similar in all cases, occurring at ~ 20 TABLE I1 Effect of Glibenclamide on CFTR Mutants 4-50 mV -90 mV Mutant Experiments Ki n Ki n my/ u2v/ CFTR 21.8 ± 5.2 0.8 - 0.1 19.0 - 5.6* 0.7 ± 0.1 7 CFTR-K335E 25.9 ± 4.1 0.9 ± 0.I 20.4 +--3.7* 0.9 ± 0.1 6 CFTR-K1250M 16.8 _ 4.2 0.8 ± 0.1 31.2 ± 2.5 1.0 - 0.1 5 Ki and n values were calculated as described in Table I for currents measured at +50 and -90 mV for the number of experiments listed. Login to comment
199 Half-maximal inhibition by glibenclamide, measured at -90 mV, showed a small but statistically significant increase in potency compared with that at +50 mV for wild-type CFTR and CFTR-K335E, but not CFFR-K1250M. Login to comment
248 Inhibition of CFTR-K335E and CFTR-K1250M CI- currents by glibenclamide resembled that of wild-type CFFR. Login to comment
249 This suggests that residues K335E and K1250M do not form a critical part of the glibenclamide interaction site. Login to comment

[hide] Defective intracellular transport and processing o... Cell. 1990 Nov 16;63(4):827-34. Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, White GA, O'Riordan CR, Smith AE
Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.
Cell. 1990 Nov 16;63(4):827-34., [PMID:1699669]

Abstract [show]
The gene associated with cystic fibrosis (CF) encodes a membrane-associated, N-linked glycoprotein called CFTR. Mutations were introduced into CFTR at residues known to be altered in CF chromosomes and in residues believed to play a role in its function. Examination of the various mutant proteins in COS-7 cells indicated that mature, fully glycosylated CFTR was absent from cells containing delta F508, delta 1507, K464M, F508R, and S5491 cDNA plasmids. Instead, an incompletely glycosylated version of the protein was detected. We propose that the mutant versions of CFTR are recognized as abnormal and remain incompletely processed in the endoplasmic reticulum where they are subsequently degraded. Since mutations with this phenotype represent at least 70% of known CF chromosomes, we argue that the molecular basis of most cystic fibrosis is the absence of mature CFTR at the correct cellular location.

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112 We also made the equivalent mutation within the second nucleotide binding domain (K1250M), and we changed to glutamine both asparagine residues (at 894 and 900) to which carbohydrate is predicted to be attached (N894,900Q) (Riordan et al., 1989). Login to comment
117 Analysis of Mutant Forms of CFTR Expression vectors containing wild-type CFTR (pMT-CFTR, lane 2) and those containing the mutants pMT-CFTR-K464M (lane 3) pMT-CFTR-K1250M (lane 4) pMT-CFTR-Al507 (lane 5) pMT-CFTR-N894. Login to comment
137 CFTR Mutants Mutant CF Exon CFTR Domain A B C Wild type R334W K464M Al507 AF508 F508R s5491 G551 D N894,900Q K1250M Tthllll Y 7 N 9 Y 10 Y 10 N 10 Y 11 Y 11 N 15 N 20 N 22 TM6 NBDl NBDl NBDl NBDl NBDl NBDl ECD4 NBD2 Term - + ++ - + ++ - + - - + - - + - - + - - + - - + ++ + - - - + ++ - + - The known association with CF (Y, yes; N, no), exon localization, domain location, and presence (+ ) or absence (- ) of bands A, B, and C of mutant CFTR species is shown. Login to comment
177 K484M does not produce mature CFTR, whereas K1250M does. Login to comment
179 We need to study the ability of these mutants to complement the chloride channel defect in CF epithelial cells to establish, for example, whether K1250M and K464M both abolish function. Login to comment