ABCMdb

ABCC7 p.Glu1371Gln

ClinVar:	c.4111G>T , p.Glu1371* D , Likely pathogenic
CF databases:	c.4111G>T , p.Glu1371* D , CF-causing
Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (80%), F: D (95%), G: D (95%), H: D (95%), I: D (95%), K: D (91%), L: D (95%), M: D (91%), N: D (91%), P: D (95%), Q: D (91%), R: D (95%), S: D (91%), T: D (91%), V: D (91%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: N, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: N, R: D, S: D, T: D, V: D, W: D, Y: D,

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Publications
[hide] CFTR channel opening by ATP-driven tight dimerizat... Nature. 2005 Feb 24;433(7028):876-80. Vergani P, Lockless SW, Nairn AC, Gadsby DC
CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains.
Nature. 2005 Feb 24;433(7028):876-80., 2005-02-24 [PMID:15729345]

Abstract [show]
ABC (ATP-binding cassette) proteins constitute a large family of membrane proteins that actively transport a broad range of substrates. Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ABC proteins in that its transmembrane domains comprise an ion channel. Opening and closing of the pore have been linked to ATP binding and hydrolysis at CFTR's two nucleotide-binding domains, NBD1 and NBD2 (see, for example, refs 1, 2). Isolated NBDs of prokaryotic ABC proteins dimerize upon binding ATP, and hydrolysis of the ATP causes dimer dissociation. Here, using single-channel recording methods on intact CFTR molecules, we directly follow opening and closing of the channel gates, and relate these occurrences to ATP-mediated events in the NBDs. We find that energetic coupling between two CFTR residues, expected to lie on opposite sides of its predicted NBD1-NBD2 dimer interface, changes in concert with channel gating status. The two monitored side chains are independent of each other in closed channels but become coupled as the channels open. The results directly link ATP-driven tight dimerization of CFTR's cytoplasmic nucleotide-binding domains to opening of the ion channel in the transmembrane domains. This establishes a molecular mechanism, involving dynamic restructuring of the NBD dimer interface, that is probably common to all members of the ABC protein superfamily.

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71 Time constants for current decay fit lines (blue): WT, t ¼ 0.45 s; E1371Q, t ¼ 476 s. Note the fivefold expanded timescale for the WT record. Login to comment
73 Evidence supporting our speculation (Fig. 1a) that the CFTR-channel open state corresponds to the dimerized NBD conformation is provided by the approximately 1,000-fold stabilization of the open-burst state that results from mutation of the possible catalytic base4,17 , Glu 1371 (a glutamate in the NBD2-head 'Walker B` motif), to Gln (E1371Q; Fig. 1b). Login to comment
74 Because CFTR channels do not open without ATP (see below), current decay upon the removal of ATP reflects channel closing, and its time course measures the open burst duration; closing was complete within about 1 s of ATP withdrawal for wild-type (WT) CFTR channels (mean open burst duration was less than 0.5 s), but had a time constant of 411 ^ 64 s (mean ^ s.e.m., n ¼ 16) for mutant E1371Q channels (Fig. 1b). 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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147 Comparison of speed of closing of wild-type CFTR (a) and of mutant CFTR bearing the single mutation E1371Q (b), of the conserved Walker B glutamate. Login to comment
149 The greatly delayed closing of all four E1371Q channels (b), compared with the four WT CFTR channels (a), after removal of ATP supports the conclusion that ATP hydrolysis at the NBD2 composite catalytic site controls normal channel closing63-66,72-77,80 , and that the channel open-burst state corresponds to NBD dimerization22,29,69,70,80 . Login to comment
150 E1371Q ATPATP WT 0.5 pA 60 s a b opened only inefficiently by millimolar concentrations of poorly hydrolysable analogues such as AMP-PNP, AMP-PCP or ATP-γS53,62-64 , and not at all by ADP62 . Login to comment
174 One clue that the CFTR-channel open state corresponds to the heterodimerized NBD1-NBD2 conformation comes from the ~1,000-fold stabilization of the open state (Fig. 4b) caused by the mutation E1371Q of the Walker B Glu (the possible catalytic base29,81 ). Login to comment

[hide] The Walker B motif of the second nucleotide-bindin... Biochem J. 2007 Jan 15;401(2):581-6. Stratford FL, Ramjeesingh M, Cheung JC, Huan LJ, Bear CE
The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1-NBD2 heterodimer.
Biochem J. 2007 Jan 15;401(2):581-6., 2007-01-15 [PMID:16989640]

Abstract [show]
CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory 'R' domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1-NBD2 heterodimer.

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6 The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. Login to comment
41 For E1371Q, the reverse primer 5 - CAAATGAGCACTGGGTTGATCAAGCAGC-3 and the forward primer 5 -GCTGCTTGATCAACCCAGTGCTCATTTG-3 were used. Login to comment
65 ATP depletion experiments Infected Sf9 cells expressing NBD1 and NBD2 (wild-type or E1371Q) were washed with PBS and collected by centrifugation. Login to comment
77 The dose-response of TNP-ATP binding to the mutant NBDs: S573E and E1371Q was analysed using a single site binding algorithm and curve fitting by GraphPad Prism to determine Kd values. Login to comment
84 Similarly, the Walker B mutant proteins (S573E) and (E1371Q) bearing polyhistidine tags and an HA tag, in the case of E1371Q, were expressed in Sf9 cells using the baculovirus system (Figure 1B). Login to comment
86 Both the purified NBD1 mutant (S573E) and purified NBD2 mutant (E1371Q) bind the ATP analogue, TNP-ATP, with low micromolar affinities (Figure 2), similar to the affinities previously reported for wild-type NBD1 (11.6 µM) and NBD2 (10.6 µM) [14]. Login to comment
88 Walker B mutant of NBD2 (E1371Q) inhibits normal ATPase activity conferred by heterodimerization with NBD1 The ATPase activity of CFTR is conferred by heterodimerization of NBD1 and NBD2. Login to comment
164 Interestingly, we could not detect a significant difference in the association between wild-type NBD1 and NBD2 and the association between NBD1 and NBD2(E1371Q) following ATP depletion in cell culture prior to cell lysis (with the removal of extracellular glucose, and the addition of deoxyglucose and rotenone). Login to comment
165 In light of the recent studies by Vergani et al. [30] showing that ATP binding induced hydrogen bond formation between NBD1 and NBD2 in the vicinity of the 'catalytic site`, we predicted that the most stable interaction may have occurred between NBD1 and NBD2- (E1371Q) in ATP-replete cells. Login to comment

[hide] Thermodynamics of CFTR channel gating: a spreading... J Gen Physiol. 2006 Nov;128(5):523-33. Epub 2006 Oct 16. Csanady L, Nairn AC, Gadsby DC
Thermodynamics of CFTR channel gating: a spreading conformational change initiates an irreversible gating cycle.
J Gen Physiol. 2006 Nov;128(5):523-33. Epub 2006 Oct 16., [PMID:17043148]

Abstract [show]
CFTR is the only ABC (ATP-binding cassette) ATPase known to be an ion channel. Studies of CFTR channel function, feasible with single-molecule resolution, therefore provide a unique glimpse of ABC transporter mechanism. CFTR channel opening and closing (after regulatory-domain phosphorylation) follows an irreversible cycle, driven by ATP binding/hydrolysis at the nucleotide-binding domains (NBD1, NBD2). Recent work suggests that formation of an NBD1/NBD2 dimer drives channel opening, and disruption of the dimer after ATP hydrolysis drives closure, but how NBD events are translated into gate movements is unclear. To elucidate conformational properties of channels on their way to opening or closing, we performed non-equilibrium thermodynamic analysis. Human CFTR channel currents were recorded at temperatures from 15 to 35 degrees C in inside-out patches excised from Xenopus oocytes. Activation enthalpies(DeltaH(double dagger)) were determined from Eyring plots. DeltaH(double dagger) was 117 +/- 6 and 69 +/- 4 kJ/mol, respectively, for opening and closure of partially phosphorylated, and 96 +/- 6 and 73 +/- 5 kJ/mol for opening and closure of highly phosphorylated wild-type (WT) channels. DeltaH(double dagger) for reversal of the channel opening step, estimated from closure of ATP hydrolysis-deficient NBD2 mutant K1250R and K1250A channels, and from unlocking of WT channels locked open with ATP+AMPPNP, was 43 +/- 2, 39 +/- 4, and 37 +/- 6 kJ/mol, respectively. Calculated upper estimates of activation free energies yielded minimum estimates of activation entropies (DeltaS(double dagger)), allowing reconstruction of the thermodynamic profile of gating, which was qualitatively similar for partially and highly phosphorylated CFTR. DeltaS(double dagger) appears large for opening but small for normal closure. The large DeltaH(double dagger) and DeltaS(double dagger) (TDeltaS(double dagger) >/= 41 kJ/mol) for opening suggest that the transition state is a strained channel molecule in which the NBDs have already dimerized, while the pore is still closed. The small DeltaS(double dagger) for normal closure is appropriate for cleavage of a single bond (ATP's beta-gamma phosphate bond), and suggests that this transition state does not require large-scale protein motion and hence precedes rehydration (disruption) of the dimer interface.

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191 Even for the Walker B Glu mutant E1371Q CFTR channels, which display the most stable open-burst state observed for CFTR (closing time constant ‫004ف‬ s; Vergani et al., 2005), corresponding to Δ maxG‡ = 88 kJ/mol, if ∆H‡ is assumed ‫04ف‬ kJ/mol (its experimental determination would be a formidable task), the model predicts α Δ O-CG = -12 kJ/ mol, and again a similar overall energy profile. Login to comment

[hide] Conformational changes in a pore-lining helix coup... J Biol Chem. 2008 Feb 22;283(8):4957-66. Epub 2007 Dec 3. Beck EJ, Yang Y, Yaemsiri S, Raghuram V
Conformational changes in a pore-lining helix coupled to cystic fibrosis transmembrane conductance regulator channel gating.
J Biol Chem. 2008 Feb 22;283(8):4957-66. Epub 2007 Dec 3., 2008-02-22 [PMID:18056267]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ATP-binding cassette transporters in that it functions as an ion channel. In CFTR, ATP binding opens the channel, and its subsequent hydrolysis causes channel closure. We studied the conformational changes in the pore-lining sixth transmembrane segment upon ATP binding by measuring state-dependent changes in accessibility of substituted cysteines to methanethiosulfonate reagents. Modification rates of three residues (resides 331, 333, and 335) near the extracellular side were 10-1000-fold slower in the open state than in the closed state. Introduction of a charged residue by chemical modification at two of these positions (resides 331 and 333) affected CFTR single-channel gating. In contrast, modifications of pore-lining residues 334 and 338 were not state-dependent. Our results suggest that ATP binding induces a modest conformational change in the sixth transmembrane segment, and this conformational change is coupled to the gating mechanism that regulates ion conduction. These results may establish a structural basis of gating involving the dynamic rearrangement of transmembrane domains necessary for vectorial transport of substrates in ATP-binding cassette transporters.

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120 Functional Effects of Cd2ϩ and MTSEA on Substituted Cysteines in E1371Q-CFTR-To study the accessibility of substituted cysteines in the open state, the effects of Cd2ϩ and MTSEA were examined for CFTR channels bearing the E1371Q mutation. Login to comment
123 Hence, the observed effects of Cd2ϩ and MTS reagents, on E1371Q CFTR represent effects on open channel state. Login to comment
124 As shown in Fig. 3A, the open probability (Po) of wild type CFTR channels was about 0.2, whereas it was nearly 1 for E1371Q-CFTR. Login to comment
137 A, effect of E1371Q mutation on CFTR channel activity. Login to comment
138 Representative single-channel current traces of WT (1371E; upper trace), and E1371Q (lower trace) CFTR. Login to comment
140 Note the time scale for E1371Q record. Login to comment
141 The mean Po of WT and E1371Q CFTR was 0.28 Ϯ 0.02 (n ϭ 7) and 0.93 Ϯ 0.02 (n ϭ 7), respectively. Login to comment
154 The differences in the magnitude of MTS modification suggest that in E1371Q background there is a significant change in TM6 structure. Login to comment
156 Effect of E1371Q Mutation on Thiol Modification Rates-To obtain quantitative information about changes in reactivity, the rates of substituted cysteine modification were calculated by fitting the time course of Cl-conductance modification with a single exponential, which was then used to calculate second order reaction rate constants for various MTS reagents. Login to comment
162 The difference in reaction rates between WT and Gln1371 channels was greatest for K335C, which reacted nearly 800 times more slowly in E1371Q background. Login to comment
164 MTSEA, and MTSES modification rates of residues in TM6 in WT and E1371Q backgrounds. Login to comment
168 CFTR Conformation Changes during Gating FEBRUARY 22, 2008•VOLUME 283•NUMBER 8 JOURNAL OF BIOLOGICAL CHEMISTRY 4961 decrease, and I331C reacted 5-fold slower (Fig. 4, B and C) in E1371Q channels. Login to comment
172 Alternatively, it is possible that the observed reaction rates in E1371Q mutational background are due to a change in the channel structure induced by the mutation and not related to structural changes associated with open channel state. Login to comment
222 State-dependent Reactivity Reflects a Local Rearrangement and Exposure of the Side Chains-We observed large differences in the rates of cysteine modification at three (residues 331, 333, and 335) of the five residues in the E1371Q background. Login to comment
232 Therefore, we cannot exclude the possibility that the combination of these two mutations, K335C and E1371Q is responsible for the observed changes in reactivity of K335C. Login to comment
247 In contrast, our results show that the reactivity of R334C does not exhibit state dependence either under varying activation levels or in the E1371Q channel. Login to comment
249 A notable difference between the two mutations is that the Walker A mutation K1250A, unlike E1371Q, decreases the ATP binding affinity of NBD2 (27), which profoundly reduces the channel opening rate (28, 29) in addition to decreasing the closing rate (30) of CFTR. Login to comment

[hide] The intact CFTR protein mediates ATPase rather tha... Biochem J. 2008 Jun 1;412(2):315-21. Ramjeesingh M, Ugwu F, Stratford FL, Huan LJ, Li C, Bear CE
The intact CFTR protein mediates ATPase rather than adenylate kinase activity.
Biochem J. 2008 Jun 1;412(2):315-21., 2008-06-01 [PMID:18241200]

Abstract [show]
The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

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5 Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Login to comment
19 The channel function of the CFTR-E1371Q mutant has been studied, and it exhibits a prolonged channel open time, consistent with the idea that ATPase activity promotes channel closure [6,7]. Login to comment
26 Furthermore, the consequences of the disease-causing mutation G551D and the catalytic base mutation E1371Q on the enzymatic activity of the full-length protein were also evaluated. Login to comment
36 Purification and reconstitution of wt (wild-type), CFTR-G551D and CFTR-E1371Q Detailed protocols regarding the generation of wt and mutant CFTR-His10 proteins are described elsewhere [29,30]. Login to comment
118 Interestingly, the addition of AMP (400 μM) to PKA-treated CFTR did not exert any significant effect on its ATPase activity (Figures 3A and 5B), a finding which is consistent with previous Figure 4 The disease-causing mutant CFTR-G551D and the putative catalytic base mutant CFTR-E1371Q exhibit reduced ATPase activity (A) Silver-stained gel showing purification of CFTR-E1371Q. Login to comment
119 (B) Phosphoimage shows Pi production by proteoliposomes containing CFTR-E1371Q (right panel) compared with empty liposomes in the absence (-) or presence (+) of 400 μM AMP. Login to comment
120 (C) ATPase activity (Results are means + - S.E.M.) of CFTR-G551D (n = 6) or CFTR-E1371Q (n = 6). Login to comment
125 The enzymatic activity of full-length mutant CFTR proteins: CFTRG551D and CFTR-E1371Q Gly551 is located in the signature motif of NBD1 [1] and is thought to exist at the interface through which NBD1 and NBD2 interact to form the catalytic site for ATP hydrolysis [4,7]. Login to comment
132 Therefore we expressed the full-length mutant protein CFTR-E1371Q and purified it from Sf9 cells (Figure 4A) for the purpose of studying its ATPase activity. Login to comment
136 As for the wt and CFTR-G551D proteins, there was no measurable adenylate kinase activity stimulated in CFTR-E1371Q by the addition of 400 μM AMP (Figure 4B). Login to comment
198 Substitution of Glu1371 with a glutamine residue has been shown to delay the rate of channel closing, providing support for the idea that ATPase activity promotes closure of the channel gate [6,7]. Login to comment
200 Interestingly, the ATPase activity of CFTR was not completely inhibited in the CFTR-E1371Q mutant, which exhibited approx. Login to comment

[hide] The H-loop in the second nucleotide-binding domain... Cell Physiol Biochem. 2010;25(2-3):169-80. Epub 2010 Jan 12. Kloch M, Milewski M, Nurowska E, Dworakowska B, Cutting GR, Dolowy K
The H-loop in the second nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator is required for efficient chloride channel closing.
Cell Physiol Biochem. 2010;25(2-3):169-80. Epub 2010 Jan 12., [PMID:20110677]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR-->AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR-->AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface.

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195 Substitution of this conserved glutamate with either serine (E1371S) or glutamine (E1371Q) produces a channel that displays prolonged open times [3, 16, 21, 59]. Login to comment
196 Importantly, a recent study has demonstrated that the E1371Q substitution leads to significant decrease ofATPase activity in the full-length CFTR [50] and an earlier study has shown that the same mutation abolishes the ATPase activity of the NBD1-NBD2 heterodimer, while not affecting the nucleotide binding affinity of NBD2 [49]. Login to comment
197 Hence, although we have no data regarding the influence of E1371Q on the CFTR opening rate, it seems that mutations affecting either the H-loop or Glu1371 have a very similar impact on ATP-dependent CFTR gating, which suggests close cooperation of both these elements, as predicted in the catalytic dyad model. Login to comment

[hide] Regulation of conductance by the number of fixed p... J Gen Physiol. 2010 Mar;135(3):229-45. Epub 2010 Feb 8. Zhou JJ, Li MS, Qi J, Linsdell P
Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore.
J Gen Physiol. 2010 Mar;135(3):229-45. Epub 2010 Feb 8., [PMID:20142516]

Abstract [show]
Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be "transplanted" to a residue in a different transmembrane (TM) region (S1141). Thus, the mutant channel K95S/S1141K showed Cl(-) conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby "rescuing" the effects of the charge-neutralizing K95S mutation. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). The S1141K mutant had similar Cl(-) conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO(2))(4)(2-) in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl(-) channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl(-) transport through a combination of failure to increase Cl(-) conductance and increased susceptibility to channel block by cytosolic substances.

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32 In some cases (see Fig. 7 and Fig. S4), macroscopic currents were recorded from cell-attached patches on unstimulated cells expressing spontaneously active E1371Q-CFTR channels. Login to comment
36 Because channels bearing the E1371Q mutation appeared to be spontaneously active even in the absence of ATP (see Fig. S4), inside-out patch recordings of this construct were made in the absence of PKA or ATP unless stated otherwise. Login to comment
37 Background current levels in these E1371Q mutants were determined at the end of the experiment by adding a high concentration (10 µM) of the specific CFTR inhibitor CFTRinh-172 (Ma et al., 2002) to the cytoplasmic solution in excised inside-out membrane patches. Login to comment
65 Fig. S4 compares the activity of wild type and E1371Q in on-cell and inside-out membrane patches. Login to comment
67 Fig. S6 shows the block of E1371Q and E1371Q/S1141K by intracellular Pt(NO2)4 2 ions. Login to comment
82 For E1371Q mutant channels, background currents were determined after the addition of 10 µM CFTRinh-172 (see above). Login to comment
87 Because of the effects of ATP on CFTR channel gating, these experiments were performed in an E1371Q background. Login to comment
193 Mean of data from four to five patches in B-D. tion of 10 mM ATP caused a potent inhibition of S1141K/E1371Q current during 400-ms hyperpolarizing voltage steps from a holding potential of +60 mV (Fig. 6 A). Login to comment
194 Time-dependent inhibition by ATP was still apparent; however, use of the E1371Q background provides direct evidence that ATP inhibits current flow because the amplitude of the current is greatly reduced. Login to comment
197 In contrast to the strong inhibition of S1141K/ E1371Q current by ATP under these conditions, 10 mM prolongation of CFTR channel open times (Vergani et al., 2003; Gadsby et al., 2006; Stratford et al., 2007). Login to comment
198 To our surprise, we found that the E1371Q mutant was constitutively active in on-cell recordings from intact BHK cells, and that even after excision into nominally ATP-free solutions, channel activity remained maximal (Fig. S4). Login to comment
199 Although the reasons for this constitutive activity, which contrasts with a complete lack of spontaneous activity we observe for other CFTR constructs expressed in BHK cells, are unknown, it did allow us to quantify ATP effects on S1141K current amplitude in an E1371Q background in inside-out membrane patches, beginning with 0 ATP control conditions (Fig. 6). Login to comment
200 With a low extracellular Cl concentration (4 mM), the addi- Figure 6. Inhibition of S1141K/E1371Q-CFTR by intracellular ATP. Login to comment
201 (A) Example macroscopic currents carried by S1141K/E1371Q during hyperpolarizing voltage steps to between +60 and 100 mV recorded under conditions of low extracellular Cl concentration (4 mM). Login to comment
205 Also shown are the effects of 10 mM ATP on E1371Q () and K95S/S1141K/E1371Q () at 100 mV. Login to comment
206 (C) Example macroscopic S1141K/E1371Q currents during voltage steps to between +60 and 100 mV recorded with a high extracellular Cl concentration (154 mM) before (control) and after the addition of 10 mM Na2ATP to the intracellular solution in the absence of PKA. Login to comment
213 We took advantage of constitutive activity of E1371Q in BHK cells (see above) to monitor channel block in intact cells. CFTR activity was monitored using a voltage step protocol, both during on-cell recording and after patch excision into the inside-out configuration in the absence of ATP and PKA (Fig. 7). Login to comment
216 With high Cl concentration pipette solution, E1371Q showed outward rectification of the macroscopic I-V relationship during cell-attached recording (Fig. 7, A and B). Login to comment
218 Outward rectification in cell-attached patches under these conditions was even more pronounced in S1141K/ E1371Q, and again this rectification was relieved by excision of the membrane patch, resulting in a linear I-V relationship in inside-out patches. Login to comment
221 Such analysis revealed that current inhibition in cell-attached patches was significantly stronger in S1141K/E1371Q than in E1371Q at hyperpolarized voltages, both at high extracellular Cl concentrations (Fig. 7 C) and at low Cl concentrations (Fig. 7 D). Login to comment
224 However, the effects of complementary mutations at K95 and at S1141 in TM12 suggest that the important functional role of this positive ATP had no effect on E1371Q and only a very small inhibitory effect on K95S/S1141K/E1371Q (Fig. 6 B). Login to comment
225 Interestingly, the inhibition of S1141K/E1371Q by intracellular ATP was very much weaker when using a high extracellular Cl concentration (154 mM; Fig. 6 C) during voltage steps of the same duration. Login to comment
237 Under ATP-free conditions, block by intracellular Pt(NO2)4 2 was dramatically more potent in E1371Q/S1141K compared with E1371Q alone, leading to a 38-fold decrease in mean Kd(0) (Fig. S6). Login to comment
240 (A) Example macroscopic currents carried by E1371Q and S1141K/E1371Q-CFTR in cell-attached patches (left panels) after excision into the inside-out patch configuration (middle panels) and after the addition of 10 µM CFTRinh-172 to the intracellular solution (right panels). Login to comment
271 This would not be surprising in evolutionary terms because maximization of Cl conductance is the physiologically meaningful role of the positive charge associated with K95 in this part of the inner vestibule of the pore, with interaction with channel blockers current amplitude in cell-attached patches as a fraction of current in the same patch after excision into the inside-out patch configuration for both E1371Q () and S1141K/E1371Q (), with 154 mM Cl (C) or 4 mM Cl (D) in the extracellular solution. Login to comment
284 Consistent with this, the apparent affinity of block by small, divalent Pt(NO2)4 2 anions was increased 38-fold in S1141K/ E1371Q compared with E1371Q (Fig. S6). Login to comment
338 As shown in Fig. 7, this inhibition is pronounced in BHK cells, leading to an 60% block of wild-type (actually E1371Q) currents in intact cells at 100 mV with high extracellular Cl . Login to comment
340 Although block appears strong in wild type (E1371Q), it is still significantly strengthened in S1141K (Fig. 7), suggesting that the number of fixed positive charges in the inner vestibule of the pore controls interactions with endogenous cytoplasmic-blocking molecules, and therefore (in intact cells) overall channel function in terms of the rate of anion efflux. Login to comment
343 This may be because the presence of an additional positive charge favors attraction of polyvalent anions into the pore, whereas the normal substrates of CFTR-mediated transport (Cl and HCO3  ) are monovalent anions. An overall decrease in channel function in intact cells is demonstrated in on-cell current recordings (Fig. 7), which show decreased Cl currents (relative to unblocked currents after patch excision to the inside-out configuration) at hyperpolarized voltages in S1141K/E1371Q relative to E1371Q alone. 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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201 Conversely, mutation of the catalytic glutamate to glutamine in NBD2, E1371Q, has previously been shown to stabilize NBD dimers by trapping ATP at the NBD-NBD interface (39). Login to comment
204 Stabilization of the putative NBD1-NBD2 dimer via the E1371Q mutation did not facilitate the trafficking of the ⌬F508 protein and had no discernible effect on the maturation of the wild type protein. Login to comment
282 The NBD-dimer stabilizing mutation, E1371Q, does not dramatically alter the trafficking of the wild type or ⌬F508 CFTR proteins when expressed transiently in HEK-293 cells. Login to comment
305 Stabilization of the NBD heterodimer by the E1371Q mutation had no discernible effect on wild type or ⌬F508 maturation (Fig. 5B). Login to comment
306 The failure of the E1371Q mutant to rescue ⌬F508 CFTR is consistent with ⌬F508 influence on the early step of NBD1 folding and that FIGURE 6. Login to comment
355 Acknowledgments-We thank David Gadsby for suggesting the use of the NBD dimer stabilizing E1371Q mutation and members of the Thomas laboratory for critical comments and helpful suggestions. Login to comment

[hide] Involvement of F1296 and N1303 of CFTR in induced-... J Gen Physiol. 2010 Oct;136(4):407-23. Szollosi A, Vergani P, Csanady L
Involvement of F1296 and N1303 of CFTR in induced-fit conformational change in response to ATP binding at NBD2.
J Gen Physiol. 2010 Oct;136(4):407-23., [PMID:20876359]

Abstract [show]
The chloride ion channel cystic fibrosis transmembrane conductance regulator (CFTR) displays a typical adenosine trisphosphate (ATP)-binding cassette (ABC) protein architecture comprising two transmembrane domains, two intracellular nucleotide-binding domains (NBDs), and a unique intracellular regulatory domain. Once phosphorylated in the regulatory domain, CFTR channels can open and close when supplied with cytosolic ATP. Despite the general agreement that formation of a head-to-tail NBD dimer drives the opening of the chloride ion pore, little is known about how ATP binding to individual NBDs promotes subsequent formation of this stable dimer. Structural studies on isolated NBDs suggest that ATP binding induces an intra-domain conformational change termed "induced fit," which is required for subsequent dimerization. We investigated the allosteric interaction between three residues within NBD2 of CFTR, F1296, N1303, and R1358, because statistical coupling analysis suggests coevolution of these positions, and because in crystal structures of ABC domains, interactions between these positions appear to be modulated by ATP binding. We expressed wild-type as well as F1296S, N1303Q, and R1358A mutant CFTR in Xenopus oocytes and studied these channels using macroscopic inside-out patch recordings. Thermodynamic mutant cycles were built on several kinetic parameters that characterize individual steps in the gating cycle, such as apparent affinities for ATP, open probabilities in the absence of ATP, open probabilities in saturating ATP in a mutant background (K1250R), which precludes ATP hydrolysis, as well as the rates of nonhydrolytic closure. Our results suggest state-dependent changes in coupling between two of the three positions (1296 and 1303) and are consistent with a model that assumes a toggle switch-like interaction pattern during the intra-NBD2 induced fit in response to ATP binding. Stabilizing interactions of F1296 and N1303 present before ATP binding are replaced by a single F1296-N1303 contact in ATP-bound states, with similar interaction partner toggling occurring during the much rarer ATP-independent spontaneous openings.

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242 Although for WT CFTR and for the nonhydrolytic mutant D1370N these two parameters are in rough agreement (Csanády et al., 2010), such comparisons have not yet been done for several other NBD2mutantsdefectiveinATPhydrolysis(e.g.,K1250R, K1250A, E1371S, and E1371Q). Login to comment

[hide] Regulation of CFTR chloride channel macroscopic co... Am J Physiol Cell Physiol. 2011 Jan;300(1):C65-74. Epub 2010 Oct 6. Li MS, Holstead RG, Wang W, Linsdell P
Regulation of CFTR chloride channel macroscopic conductance by extracellular bicarbonate.
Am J Physiol Cell Physiol. 2011 Jan;300(1):C65-74. Epub 2010 Oct 6., [PMID:20926782]

Abstract [show]
The CFTR contributes to Cl and HCO transport across epithelial cell apical membranes. The extracellular face of CFTR is exposed to varying concentrations of Cl and HCO in epithelial tissues, and there is evidence that CFTR is sensitive to changes in extracellular anion concentrations. Here we present functional evidence that extracellular Cl and HCO regulate anion conduction in open CFTR channels. Using cell-attached and inside-out patch-clamp recordings from constitutively active mutant E1371Q-CFTR channels, we show that voltage-dependent inhibition of CFTR currents in intact cells is significantly stronger when the extracellular solution contains HCO than when it contains Cl. This difference appears to reflect differences in the ability of extracellular HCO and Cl to interact with and repel intracellular blocking anions from the pore. Strong block by endogenous cytosolic anions leading to reduced CFTR channel currents in intact cells occurs at physiologically relevant HCO concentrations and membrane potentials and can result in up to approximately 50% inhibition of current amplitude. We propose that channel block by cytosolic anions is a previously unrecognized, physiologically relevant mechanism of channel regulation that confers on CFTR channels sensitivity to different anions in the extracellular fluid. We further suggest that this anion sensitivity represents a feedback mechanism by which CFTR-dependent anion secretion could be regulated by the composition of the secretions themselves. Implications for the mechanism and regulation of CFTR-dependent secretion in epithelial tissues are discussed.

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10 Using cell-attached and inside-out patch-clamp recordings from constitutively active mutant E1371Q-CFTR channels, we show that voltage-dependent inhibition of CFTR currents in intact cells is significantly stronger when the extracellular solution contains HCO3 - than when it contains Cl- . Login to comment
30 In the present study, we have taken advantage of the fact that mutant E1371Q-CFTR channels show constitutive, high levels of activity in baby hamster kidney (BHK) cells (61) to monitor and investigate voltage-dependent channel block in cell-attached patches. Login to comment
38 All experiments were carried out in an E1371Q-CFTR background, the properties of which were described in detail recently (61). Login to comment
40 When expressed in BHK cells, E1371Q-CFTR forms constitutively active channels that are insensitive to stimulation by exogenous protein kinase A, suggesting that the activity of these channels is Fig. 1. Anion-sensitive channel inhibition in intact cells. Login to comment
41 A and B: macroscopic currents carried by E1371Q-CFTR during depolarizing voltage ramps from -100 to ϩ60 mV, with Cl- - or gluconate-containing pipette solutions. Login to comment
51 Consistent with this, E1371Q and other mutations constructed in this background gave stable current amplitudes in cell-attached membrane patches and in excised, inside-out membrane patches under all ionic conditions investigated. Login to comment
52 Patch-clamp recordings of E1371Q-CFTR channel macroscopic currents were as described in detail recently (61). Login to comment
64 Under these conditions, changes in current amplitude and reversal potential appeared reversible (see Fig. 5), consistent with stable E1371Q channel activity over time. Login to comment
74 A: macroscopic currents carried by E1371Q-CFTR during depolarizing voltage ramps from -100 to ϩ60 mV with HCO3 - -containing pipette solution. Currents were recorded from cell-attached patches and immediately following patch excision to the inside-out configuration and following addition of 20 ␮M CFTRinh-172 to intracellular solution. Zero current level is indicated by dotted line. B: macroscopic I-V relationships for patch described in A following subtraction of leak current recorded in the presence of CFTRinh-172 in on-cell and inside-out patch configurations. Login to comment
84 Recently, we used constitutively active E1371Q-CFTR channels expressed in BHK cells to quantify the degree of channel inhibition that underlies this outward rectification in intact cells (61). Login to comment
91 A: leak-subtracted E1371Q-CFTR macroscopic I-V relationships recorded with Cl- -, gluconate-, or HCO3 - -containing pipette solutions. Login to comment
105 This confirms that gluconate and HCO3 - are less able to generate knock off of intracellular open channel blockers in E1371Q-CFTR than is Cl- . Login to comment
112 Interestingly, PHCO3/PCl for E1371Q-CFTR estimated using a high concentration of extracellular HCO3 - (0.12 Ϯ 0.01, n ϭ 12; Fig. 4D) was significantly lower (P Ͻ 0.02) than that under the reversed ionic gradient (150 mM internal HCO3 - , 154 mM external Cl- ; Fig. 5). Login to comment
118 To investigate whether these positively charged residues influence the strength of channel block by cytosolic anions in intact cells, we neutralized these charges in an E1371Q background. Login to comment
119 Figure 6A shows macroscopic currents carried by K95Q/ E1371Q-, R303Q/E1371Q-, and K978Q/E1371Q-CFTR in Fig. 4. Login to comment
130 Comparison of the fractional current in cell-attached patches seen in these mutants with those for E1371Q under the same ionic conditions (Fig. 1) suggests that block of each of these mutants is weakened under cell-attached conditions, particularly at hyperpolarized voltages, where block is usually strongest (Fig. 6B). Login to comment
131 Interestingly, the effects of the K95Q and R303Q mutations did not appear additive, with the K95Q/R303Q/E1371Q mutant showing relief of block following patch excision similar to that shown by K95Q/E1371Q (Fig. 6). Login to comment
132 Although channels bearing the K95Q mutation did give smaller currents in cell-attached patches than those seen following patch excision, this effect appeared voltage-independent, in contrast to the clearly voltage-dependent inhibition of E1371Q (Fig. 6B). Login to comment
144 Permeability of intracellular HCO3 - in E1371Q-CFTR. Login to comment
145 A: macroscopic currents carried by E1371Q in an inside-out patch during depolarizing voltage ramps from -100 to ϩ60 mV with Cl- -containing pipette solution. Currents were recorded after they had attained a stable amplitude in inside-out patches, during perfusion with Cl- - or HCO3 - -containing intracellular (bath) solutions and following addition of 20 ␮M CFTRinh-172 to intracellular solution. Zero current level is indicated by dotted line. B: macroscopic I-V relationships for patch in A following subtraction of leak current recorded in the presence of CFTRinh-172, when intracellular solution contained Cl- or HCO3 - . Login to comment
151 In native epithelial cells, we would expect changes in external [Cl- ] and [HCO3 - ] to alter open channel conductance (present study) and channel open probability (38, 58); the use of the constitutively active, gating-defective E1371Q-CFTR mutant in the present study was therefore important to isolate and characterize anion effects on open channels. Login to comment
154 A: macroscopic I-V relationships for channel mutants (in an E1371Q background) following subtraction of leak currents recorded in the presence of CFTRinh-172 in on-cell and inside-out patch configurations with gluconate-containing pipette solutions. Login to comment
155 B: quantification of current inhibition in intact cells. Macroscopic current amplitude in cell-attached patches is plotted as a fraction of current in the same patch immediately after excision to inside-out patch configuration for the named mutant () and for E1371Q as control (Œ, see Fig. 1). Login to comment
157 Values are means for 18 (for E1371Q) and 5-7 (for other mutants) patches. Login to comment
158 Significant difference from E1371Q: *P Ͻ 0.02; **P Ͻ 0.00001. Login to comment
173 The HCO3 - permeability of E1371Q-CFTR (Fig. 4) was comparable to that reported many times previously for wild-type CFTR in epithelial cells and heterologous expression systems (25, 55). Login to comment
9 Using cell-attached and inside-out patch-clamp recordings from constitutively active mutant E1371Q-CFTR channels, we show that voltage-dependent inhibition of CFTR currents in intact cells is significantly stronger when the extracellular solution contains HCO3 - than when it contains Cl- . Login to comment
29 In the present study, we have taken advantage of the fact that mutant E1371Q-CFTR channels show constitutive, high levels of activity in baby hamster kidney (BHK) cells (61) to monitor and investigate voltage-dependent channel block in cell-attached patches. Login to comment
37 All experiments were carried out in an E1371Q-CFTR background, the properties of which were described in detail recently (61). Login to comment
39 When expressed in BHK cells, E1371Q-CFTR forms constitutively active channels that are insensitive to stimulation by exogenous protein kinase A, suggesting that the activity of these channels is Fig. 1. Anion-sensitive channel inhibition in intact cells. Login to comment
50 Consistent with this, E1371Q and other mutations constructed in this background gave stable current amplitudes in cell-attached membrane patches and in excised, inside-out membrane patches under all ionic conditions investigated. Login to comment
63 Under these conditions, changes in current amplitude and reversal potential appeared reversible (see Fig. 5), consistent with stable E1371Q channel activity over time. Login to comment
73 A: macroscopic currents carried by E1371Q-CFTR during depolarizing voltage ramps from -100 to ϩ60 mV with HCO3 - -containing pipette solution. Currents were recorded from cell-attached patches and immediately following patch excision to the inside-out configuration and following addition of 20 ␮M CFTRinh-172 to intracellular solution. Zero current level is indicated by dotted line. B: macroscopic I-V relationships for patch described in A following subtraction of leak current recorded in the presence of CFTRinh-172 in on-cell and inside-out patch configurations. Login to comment
83 Recently, we used constitutively active E1371Q-CFTR channels expressed in BHK cells to quantify the degree of channel inhibition that underlies this outward rectification in intact cells (61). Login to comment
90 A: leak-subtracted E1371Q-CFTR macroscopic I-V relationships recorded with Cl- -, gluconate-, or HCO3 - -containing pipette solutions. Login to comment
104 This confirms that gluconate and HCO3 - are less able to generate knock off of intracellular open channel blockers in E1371Q-CFTR than is Cl- . Login to comment
111 Interestingly, PHCO3/PCl for E1371Q-CFTR estimated using a high concentration of extracellular HCO3 - (0.12 Ϯ 0.01, n ϭ 12; Fig. 4D) was significantly lower (P Ͻ 0.02) than that under the reversed ionic gradient (150 mM internal HCO3 - , 154 mM external Cl- ; Fig. 5). Login to comment
117 To investigate whether these positively charged residues influence the strength of channel block by cytosolic anions in intact cells, we neutralized these charges in an E1371Q background. Login to comment
129 Comparison of the fractional current in cell-attached patches seen in these mutants with those for E1371Q under the same ionic conditions (Fig. 1) suggests that block of each of these mutants is weakened under cell-attached conditions, particularly at hyperpolarized voltages, where block is usually strongest (Fig. 6B). Login to comment
143 Permeability of intracellular HCO3 - in E1371Q-CFTR. Login to comment
150 In native epithelial cells, we would expect changes in external [Cl- ] and [HCO3 - ] to alter open channel conductance (present study) and channel open probability (38, 58); the use of the constitutively active, gating-defective E1371Q-CFTR mutant in the present study was therefore important to isolate and characterize anion effects on open channels. Login to comment
153 A: macroscopic I-V relationships for channel mutants (in an E1371Q background) following subtraction of leak currents recorded in the presence of CFTRinh-172 in on-cell and inside-out patch configurations with gluconate-containing pipette solutions. Login to comment
156 Values are means for 18 (for E1371Q) and 5-7 (for other mutants) patches. Login to comment
172 The HCO3 - permeability of E1371Q-CFTR (Fig. 4) was comparable to that reported many times previously for wild-type CFTR in epithelial cells and heterologous expression systems (25, 55). Login to comment

[hide] Application of high-resolution single-channel reco... Methods Mol Biol. 2011;741:419-41. Cai Z, Sohma Y, Bompadre SG, Sheppard DN, Hwang TC
Application of high-resolution single-channel recording to functional studies of cystic fibrosis mutants.
Methods Mol Biol. 2011;741:419-41., [PMID:21594800]

Abstract [show]
The patch-clamp technique is a powerful and versatile method to investigate the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, its malfunction in disease and modulation by small molecules. Here, we discuss how the molecular behaviour of CFTR is investigated using high-resolution single-channel recording and kinetic analyses of channel gating. We review methods used to quantify how cystic fibrosis (CF) mutants perturb the biophysical properties and regulation of CFTR. By explaining the relationship between macroscopic and single-channel currents, we demonstrate how single-channel data provide molecular explanations for changes in CFTR-mediated transepithelial ion transport elicited by CF mutants.

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202 Furthermore, relaxation analysis of macroscopic currents is the method of choice to analyse the burst duration of CFTR constructs that open for tens of hundreds of seconds (e.g. E1371Q, (47)), because it can be very difficult to collect sufficient gating transitions for microscopic kinetic analysis of channel gating for these CFTR constructs. Login to comment

[hide] Electrophysiological, biochemical, and bioinformat... Methods Mol Biol. 2011;741:443-69. Csanady L, Vergani P, Gulyas-Kovacs A, Gadsby DC
Electrophysiological, biochemical, and bioinformatic methods for studying CFTR channel gating and its regulation.
Methods Mol Biol. 2011;741:443-69., [PMID:21594801]

Abstract [show]
CFTR is the only member of the ABC (ATP-binding cassette) protein superfamily known to function as an ion channel. Most other ABC proteins are ATP-driven transporters, in which a cycle of ATP binding and hydrolysis, at intracellular nucleotide binding domains (NBDs), powers uphill substrate translocation across the membrane. In CFTR, this same ATP-driven cycle opens and closes a transmembrane pore through which chloride ions flow rapidly down their electrochemical gradient. Detailed analysis of the pattern of gating of CFTR channels thus offers the opportunity to learn about mechanisms of function not only of CFTR channels but also of their ABC transporter ancestors. In addition, CFTR channel gating is subject to complex regulation by kinase-mediated phosphorylation at multiple consensus sites in a cytoplasmic regulatory domain that is unique to CFTR. Here we offer a practical guide to extract useful information about the mechanisms that control opening and closing of CFTR channels: on how to plan (including information obtained from analysis of multiple sequence alignments), carry out, and analyze electrophysiological and biochemical experiments, as well as on how to circumvent potential pitfalls.

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135 For instance, mutations might simultaneously alter the rates of both gating transitions by up to 1000-fold with no large effect on Po (e.g., E1371Q; (1)), emphasizing the importance of kinetic analysis for mechanistic understanding. Login to comment

[hide] Pseudohalide anions reveal a novel extracellular s... Br J Pharmacol. 2012 Nov;167(5):1062-75. doi: 10.1111/j.1476-5381.2012.02041.x. Li MS, Cowley EA, Linsdell P
Pseudohalide anions reveal a novel extracellular site for potentiators to increase CFTR function.
Br J Pharmacol. 2012 Nov;167(5):1062-75. doi: 10.1111/j.1476-5381.2012.02041.x., [PMID:22612315]

Abstract [show]
BACKGROUND AND PURPOSE There is great interest in the development of potentiator drugs to increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis. We tested the ability of several anions to potentiate CFTR activity by a novel mechanism. EXPERIMENTAL APPROACH Patch clamp recordings were used to investigate the ability of extracellular pseudohalide anions (Co(CN)(6) (3-) , Co(NO(2) )(6) (3-) , Fe(CN)(6) (3-) , IrCl(6) (3-) , Fe(CN)(6) (4-) ) to increase the macroscopic conductance of mutant CFTR in intact cells via interactions with cytoplasmic blocking anions. Mutagenesis of CFTR was used to identify a possible molecular mechanism of action. Transepithelial short-circuit current recordings from human airway epithelial cells were used to determine effects on net anion secretion. KEY RESULTS Extracellular pseudohalide anions were able to increase CFTR conductance in intact cells, as well as increase anion secretion in airway epithelial cells. This effect appears to reflect the interaction of these substances with a site on the extracellular face of the CFTR protein. CONCLUSIONS AND IMPLICATIONS Our results identify pseudohalide anions as increasing CFTR function by a previously undescribed molecular mechanism that involves an interaction with an extracellular site on the CFTR protein. Future drugs could utilize this mechanism to increase CFTR activity in cystic fibrosis, possibly in conjunction with known intracellularly-active potentiators.

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36 Macroscopic current recordings were carried out in an E1371Q-CFTR background (see below). Login to comment
40 Patch clamp recording of CFTR In order to monitor and compare CFTR macroscopic conductance in intact cells and in excised membrane patches, we used mutant E1371Q-CFTR channels that show constitutive, high levels of activity when expressed in BHK cells (Zhou et al., 2010; Li et al., 2011). Login to comment
42 Patch clamp recording of E1371Q-CFTR macroscopic currents was carried out as described in detail recently (Zhou et al., 2010; Li et al., 2011). Login to comment
77 Experimentally, these regulatory effects of extracellular anions can be monitored and quantified using the constitutively active E1371Q-CFTR mutant, as described in detail previously (Li et al., 2011). Login to comment
78 With low extracellular Cl- concentrations, E1371Q-CFTR currents in intact cells are small due to block by cytosolic anions, the extent of which can be observed by the increase in current amplitude due to relief of block when the membrane patch is excised to the inside-out configuration (Li et al., 2011). Login to comment
94 However, with 10 mM Co(NO2)6 3- in the extracellular Figure 1 Effect of external pseudohalide anions on macroscopic E1371Q-CFTR currents in cell-attached and inside-out membrane patches recorded with low extracellular chloride concentration. Login to comment
95 Example leak-subtracted macroscopic I-V relationships for E1371Q-CFTR recorded under low (4 mM) extracellular Cl-concentration conditions. Constitutively active currents were recorded from cell-attached patches (on cell) and immediately after patch excision (inside out). Login to comment
102 To gain information on the molecular mechanism of action of extracellular pseudohalide anions, we tested their ability to stimulate CFTR conductance in E1371Q-CFTR channels in which these positively charged residues had been neutralized. Login to comment
104 Examples of the macroscopic I-V relationships for R334Q, K892Q, R899Q and K892Q/R899Q (all in an E1371Q background) are shown in Figure 5A. Login to comment
112 Neutralization of both of these extracellular positive charges, in the K892Q/R899Q/ E1371Q triple mutant, resulted in apparent blocker sensitivity that, as in R899Q/E1371Q, was not significantly affected by extracellular Co(CN)6 3- or Co(NO2)6 3- (Figure 5D,E). Login to comment
135 (A) Example leak-subtracted macroscopic I-V relationships for E1371Q-CFTR recorded under high (154 mM) extracellular Cl-concentration conditions. Constitutively active currents were recorded from cell-attached patches (on cell) and immediately after patch excision (inside out). Login to comment
144 (A-C) Examples of leak-subtracted macroscopic I-V relationships for different mutants in an E1371Q-CFTR background recorded under low (4 mM) extracellular Cl-concentration conditions (compare E1371Q alone in Figure 1). Login to comment
157 Evidence that multivalent pseudohalide anions can increase CFTR function comes from the ability of these compounds to increase apparent macroscopic conductance of the constitutively active E1371Q-CFTR mutant in intact cells. Login to comment
158 Previously, we showed that extracellular Cl- has a similar stimulating effect on E1371Q-CFTR conductance in intact cells, via its ability to destabilize the blockage of open channels by cytosolic substances, blockage which acts to decrease CFTR conductance in a voltage-dependent fashion (Li et al., 2011). Login to comment
162 Mean fractional current recorded in cell-attached patches relative to inside-out patches for each of the channel variants named (in each case, in an E1371Q background), under low (4 mM) or high (154 mM) extracellular Cl-concentration conditions. Login to comment
170 Currently, the mechanism by which these compounds stimulate anion secretion in Calu-3 cell monolayers, and its relationship to their effect on E1371Q-CFTR macroscopic conductance in BHK cells, are not known. Login to comment
180 suggesting they might have effects on these epithelial cells unrelated to their effects on E1371Q-CFTR channels. Login to comment

[hide] CFTR: An ion channel with a transporter-type energ... J Gen Physiol. 2012 Oct;140(4):343-5. Epub 2012 Sep 10. Tsai MF
CFTR: An ion channel with a transporter-type energy-coupling mechanism.
J Gen Physiol. 2012 Oct;140(4):343-5. Epub 2012 Sep 10., [PMID:22966013]

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53 Indeed, unlike ABC transporters, which absolutely require ATPase activity to move substrates, CFTR mutants incapable of catalyzing ATP hydrolysis (e.g., K1250A, D13710N, and E1371Q) exhibit gating transitions with open probabilities comparable to WT (Powe et al., 2002; Vergani et al., 2003). Login to comment
51 Indeed, unlike ABC transporters, which absolutely require ATPase activity to move substrates, CFTR mutants incapable of catalyzing ATP hydrolysis (e.g., K1250A, D13710N, and E1371Q) exhibit gating transitions with open probabilities comparable to WT (Powe et al., 2002; Vergani et al., 2003). Login to comment

[hide] Locating a Plausible Binding Site for an Open Chan... Mol Pharmacol. 2012 Aug 24. Norimatsu Y, Ivetac A, Alexander C, O'Donnell N, Frye L, Sansom MS, Dawson DC
Locating a Plausible Binding Site for an Open Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator.
Mol Pharmacol. 2012 Aug 24., [PMID:22923500]

Abstract [show]
High-throughput screening has led to the identification of small-molecule blockers of the CFTR chloride channel, but the structural basis of blocker binding remains to be defined. We recently developed molecular models of the CFTR channel based on homology to the bacterial transporter, Sav1866, that could permit blocker binding to be analyzed in silico. The models accurately predicted the existence of a narrow region in the pore that is a likely candidate for the binding site of an open-channel pore blocker like GlyH-101, thought to act by entering the channel from the extracellular side. As a more stringent test of predictions of the CFTR pore model, we applied induced-fit, virtual ligand docking techniques to identify potential binding sites for GlyH-101 within the CFTR pore. The highest scoring, docked position was near two pore-lining residues, F337 and T338, and the rate of reaction of anionic thiol-directed reagents with cysteines substituted at either of these positions was slowed in the presence of the blocker, consistent with the predicted repulsive effect of the net negative charge on GlyH-101. When a bulky phenylalanine that forms part of the predicted binding pocket (F342) was replaced with alanine, the apparent affinity of the blocker increased by approximately 200 fold. A Molecular Mechanics-Generalized Born/Surface Area (MM-GB/SA) analysis of GlyH-101 binding predicted that substitution of F342 with alanine would substantially increase blocker affinity, primarily due to decreased intramolecular strain within the blocker-protein complex. This study suggests that GlyH-101 blocks the CFTR channel by binding within the pore bottleneck.

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220 The state-dependent reactivity of T338C CFTR observed in the current study is consistent with the finding of Beck et al., (2008) that MTSES- reacts slightly faster with a high open probability mutant T338C/E1371Q CFTR than with T338C/wt CFTR.2 Mornon et al., (2009) created a homology model of CFTR based on the inward-facing conformation of a prokaryotic transporter, MsbA (Ward et al., 2007) (PDB code: 3B5X). Login to comment
346 The state-dependent reactivity of the T338C CFTR that was observed in the current study is consistent with the finding by Beck et al. (2008) that MTSESafa; reacted slightly faster with a mutant with high open probability (T338C/ E1371Q CFTR) than with the T338C/wt CFTR.2 Mornon et al. (2009) created a homology model of the CFTR that was based on the inward-facing conformation of the prokaryotic transporter MsbA (PDB code 3B5X) (Ward et al., 2007). Login to comment
352 Wang and Linsdell (2012) studied reactions of the T338C/E1371Q CFTR with MTSESafa; and [Au(CN)2]afa; and suggested that the reaction of an engineered cysteine at position 338 with externally applied reagents was favored in the closed state. Login to comment
353 However, this observation conflicts with that by Beck et al. (2008), who reported that MTSESafa; reacted slightly faster with a double-mutant with high open probability (T338C/E1371Q CFTR) than with the T338C/wt CFTR. Login to comment
354 We observed reaction rates for MTSESafa; with T338C/wt and T338C/E1371Q CFTRs that were similar to those observed by Beck et al. (2008) (data not shown), which supports the idea that the T338C CFTR reacts in the open state. 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
58 In some experiments, channels bearing the NBD2 mutation E1371Q were used. Login to comment
69 As described above, channels bearing the E1371Q mutation were constitutively active, and whole cell currents carried by such channels were not further increased in amplitude by application of cAMP mixture, although they were sensitive to GlyH-101 (see supplemental Fig. S2). 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
90 As in our previous work (22), the effects of the E1371Q mutation were mimicked by locking channels open by treatment with 2 mM pyrophosphate (data not shown; ϳ3.9-fold increase for T338C and ϳ1.6-fold increase for L102C). 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
98 Expression of all CFTR constructs (except those containing the E1371Q mutation, see below) in baby hamster kidney cells led to the appearance of cAMP-activated whole cell currents that were inhibited by the specific CFTR inhibitor GlyH-101 (Fig. 3 and supplemental Fig. S2) and which were not observed in cells transfected with vector alone (supplemental Fig. S2). 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
111 Note that, because MTSES modification was so slow in T338C/E1371Q, the rate constant for modification of this construct was calculated from experiments using a higher concentration of MTSES (200 ␮M). 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
115 As with extracellular MTSES modification of T338C/E1371Q (Fig. 4), the rate constant for Au(CN)2 - modification of E1371Q channels was calculated from experiments using higher concentrations of Au(CN)2 - (100 ␮M). 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
146 The rate of modification of L333C and K335C, also at the extracellular end of TM6, was also decreased in an E1371Q background, suggesting slower modification of open, compared with closed channels (26). Login to comment
152 L102C, like T338C, becomes apparently more accessible to internal cysteine reactive reagents in open channels (Fig. 6B), but is inaccessible to extracellular MTSES (Fig. FIGURE 4. Login to comment
157 Modification rate constant for T338C/E1371Q was quantified from experiments using a higher concentration of MTSES (200 ␮M). Login to comment
163 One apparent problem with this explanation is that a residue only slightly closer to the outer end of TM1 (R104C) is accessible to extracellular, but not intracellular MTS reagents (see above); the model shown in Fig. 7A should put this residue close to Thr-338. Login to comment
174 Modification rate constants for T338C/E1371Q and L102C/E1371Q were quantified from experiments using a higher concentration of Au(CN)2 - (100 ␮M). 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
199 The dramatic decrease in modification rate for external MTSES and Au(CN)2 - seen in T338C/E1371Q (Figs. 4B, 5C, and 6) suggests that access to this narrow region from the extracellular solution is greatly decreased in open channels. 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
60 In some experiments, channels bearing the NBD2 mutation E1371Q were used. Login to comment
71 As described above, channels bearing the E1371Q mutation were constitutively active, and whole cell currents carried by such channels were not further increased in amplitude by application of cAMP mixture, although they were sensitive to GlyH-101 (see supplemental Fig. S2). 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
95 As in our previous work (22), the effects of the E1371Q mutation were mimicked by locking channels open by treatment with 2 mM pyrophosphate (data not shown; b03;3.9-fold increase for T338C and b03;1.6-fold increase for L102C). Login to comment
103 Expression of all CFTR constructs (except those containing the E1371Q mutation, see below) in baby hamster kidney cells led to the appearance of cAMP-activated whole cell currents that were inhibited by the specific CFTR inhibitor GlyH-101 (Fig. 3 and supplemental Fig. S2) and which were not observed in cells transfected with vector alone (supplemental Fig. S2). Login to comment
117 Note that, because MTSES modification was so slow in T338C/E1371Q, the rate constant for modification of this construct was calculated from experiments using a higher concentration of MTSES (200 òe;M). Login to comment
121 As with extracellular MTSES modification of T338C/E1371Q (Fig. 4), the rate constant for Au(CN)2 afa; modification of E1371Q channels was calculated from experiments using higher concentrations of Au(CN)2 afa; (100 òe;M). 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
181 Modification rate constants for T338C/E1371Q and L102C/E1371Q were quantified from experiments using a higher concentration of Au(CN)2 afa; (100 òe;M). 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
206 The dramatic decrease in modification rate for external MTSES and Au(CN)2 afa; seen in T338C/E1371Q (Figs. 4B, 5C, and 6) suggests that access to this narrow region from the extracellular solution is greatly decreased in open channels. 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
44 Both wild type CFTR and a variant in which all 18 endogenous cysteine residues have been substituted by other inside-out inside-out + ATP and PKA + ATP and PKA B) Q98C + MTSES (6 s) + MTSES (6 s) + MTSES (30 s) + MTSES (30 s) + MTSES (120 s) + MTSES (120 s) C) Q98C/E1371Q 200 pA 500 ms 250 pA 500 ms A) Voltage Protocol -50 -25 0 25 50 V (mV) 500 ms Fig. 1. Login to comment
48 (C) Raw currents carried by Q98C/E1371Q under these same conditions. Login to comment
49 Note that background (leak) currents are small in Q98C prior to addition of ATP and PKA, whereas in Q98C/E1371Q, as with all constructs bearing the E1371Q mutation, large constitutive currents are observed and are not further increased in amplitude by PKA and ATP (see also Refs. 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
63 In some experiments, CFTR channels bearing the NBD2 mutation E1371Q were used. This mutation results in constitutive, high levels of activity when expressed in BHK cells [23,24] (see Fig. 1C). 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
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
109 Constructs including the E1371Q mutation gave large, spontaneously active currents in BHK cells (Fig. 1C), as described previously, and likely have an open probability close to one [23,24]. Login to comment
112 Conversely, the E1371Q mutation significantly increased the rate of MTSES modification at Q98C and I344C (3.0-3.1-fold increase in modification rate constant; Pb0.02) but had no effect on the rate of modification at K95C or V345C (P>0.25) (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
158 Conversely, the E1371Q mutation significantly increased the rate of Au(CN)2 - modification at Q98C and I344C (2.8-3.5-fold increase in modification rate constant; Pb0.01) but had no effect on the rate of modification at K95C (P>0.2) (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
172 The very high apparent open probability of E1371Q mutant channels in BHK cells [23,24] means that constructs bearing this mutation likely give a good estimate of the rate of modification of open channels. Login to comment

[hide] Structural basis for the channel function of a deg... J Gen Physiol. 2011 Nov;138(5):495-507. Bai Y, Li M, Hwang TC
Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7).
J Gen Physiol. 2011 Nov;138(5):495-507., [PMID:22042986]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, but little is known about how this ion channel that harbors an uninterrupted ion permeation pathway evolves from a transporter that works by alternately exposing its substrate conduit to the two sides of the membrane. Here, we assessed reactivity of intracellularly applied thiol-specific probes with cysteine residues substituted into the 12th transmembrane segment (TM12) of CFTR. Our experimental data showing high reaction rates of substituted cysteines toward the probes, strong blocker protection of cysteines against reaction, and reaction-induced alterations in channel conductance support the idea that TM12 of CFTR contributes to the lining of the ion permeation pathway. Together with previous work, these findings raise the possibility that pore-lining elements of CFTR involve structural components resembling those that form the substrate translocation pathway of ABC transporters. In addition, comparison of reaction rates in the open and closed states of the CFTR channel leads us to propose that upon channel opening, the wide cytoplasmic vestibule tightens and the pore-lining TM12 rotates along its helical axis. This simple model for gating conformational changes in the inner pore domain of CFTR argues that the gating transition of CFTR and the transport cycle of ABC proteins share analogous conformational changes. Collectively, our data corroborate the popular hypothesis that degradation of the cytoplasmic-side gate turned an ABC transporter into the CFTR channel.

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63 Fig. S5 shows the data for modification rates in the cysless/ E1371Q background. Login to comment
203 Given that the E1371Q mutant channel has an open probability of near unity in the presence of ATP, this result suggests that 1141C is readily accessible to MTSES but not to Texas red MTSEA+ in an open channel. Similar results were observed for cysteines substituted into positions 1148, 344, and 348 under the E1371Q background. Login to comment
224 To better assess the modification rate of an open channel, we introduced an additional E1371Q mutation, which abolishes ATP hydrolysis and thereby confers an open probability of 1 on the CFTR channel. Login to comment
225 Under the E1371Q background, MTSES readily modified 1141C resulting in a decrease of current (Fig. 6 D, red line), but Texas red MTSEA+ did not react with 1141C because it neither substantially decreased the macroscopic current (Fig. 6 D, orange line) nor prevented 1141C from subsequent modification by MTSES . Login to comment

[hide] Molecular basis for the ATPase activity of CFTR. Arch Biochem Biophys. 2008 Aug 1;476(1):95-100. Epub 2008 Apr 8. Cheung JC, Kim Chiaw P, Pasyk S, Bear CE
Molecular basis for the ATPase activity of CFTR.
Arch Biochem Biophys. 2008 Aug 1;476(1):95-100. Epub 2008 Apr 8., [PMID:18417076]

Abstract [show]
CFTR is a member of the ABC (ATP binding cassette) superfamily of transporters. It is a multidomain membrane protein, which utilizes ATP to regulate the flux of its substrate through the membrane. CFTR is distinct in that it functions as a channel and it possesses a unique regulatory R domain. There has been significant progress in understanding the molecular basis for CFTR activity as an ATPase. The dimeric complex of NBD structures seen in prokaryotic ABC transporters, together with the structure of an isolated CF-NBD1, provide a unifying molecular template to model the structural basis for the ATPase activity of CFTR. The dynamic nature of the interaction between the NBDs and the R domain has been revealed in NMR studies. On the other hand, understanding the mechanisms mediating the transmission of information from the cytosolic domains to the membrane and the channel gate of CFTR remains a central challenge.

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91 We recently determined that the mutant E1371Q-CFTR retained approximately 30% of the wild type activity [34]. Login to comment
124 For example, mutations of residues in the catalytic site (Site A, Fig. 1) that decrease ATPase activity, such as mutation of the Walker A lysine residue in NBD2 (K1250A) [29] and mutation of the putative catalytic base: E1371Q leads to a decrease in the rate of channel closing [51,52,55]. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Cold Spring Harb Perspect Med. 2013 Feb 1;3(2):a009514. doi: 10.1101/cshperspect.a009514. Hunt JF, Wang C, Ford RC
Cystic fibrosis transmembrane conductance regulator (ABCC7) structure.
Cold Spring Harb Perspect Med. 2013 Feb 1;3(2):a009514. doi: 10.1101/cshperspect.a009514., [PMID:23378596]

Abstract [show]
Structural studies of the cystic fibrosis transmembrane conductance regulator (CFTR) are reviewed. Like many membrane proteins, full-length CFTR has proven to be difficult to express and purify, hence much of the structural data available is for the more tractable, independently expressed soluble domains. Therefore, this chapter covers structural data for individual CFTR domains in addition to the sparser data available for the full-length protein. To set the context for these studies, we will start by reviewing structural information on model proteins from the ATP-binding cassette (ABC) transporter superfamily, to which CFTR belongs.

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118 One key observation supporting this model is that electrophysiological studies show that the E-to-Q mutation in NBD2 of CFTR (E1371Q) produces a very long-lived open state of the channel in the presence of ATP (Vergani et al. 2005; Gadsby et al. 2006; Hwang et al. 2009). Login to comment
295 Electrophysiological studies of the H1402A and E1371Q mutations in intact human CFTR support these inferences concerning catalytic geometry by showing that the H1402A (Kloch et al. 2010) and E1371Q (Vergani et al. 2005) mutations both greatly increase the lifetime of the open state of the CFTR chloride channel, presumably because they block ATP hydrolysis and stabilize ATP-sandwich heterodimer formed by hNBD1 and hNBD2. Login to comment
297 However, despite their similar influence on the gating properties of intact CFTR, the H1402A and E1371Q mutations have dramatically different effects on the stabilityand yield of hNBD2 during purification, with the first greatly improving yield compared to the second (X Zhao, S Atwell, JF Hunt, et al., unpubl.). Login to comment
298 In silico energetic calculations suggest that the H1402A mutation stabilizes isolated hNBD2 bound to Mg-ATP, while the E1371Q mutation destabilizes it (P Kumar, C Wang, JF Hunt, et al., unpubl.). Login to comment

[hide] Cysteine scanning of CFTR's first transmembrane se... Biophys J. 2013 Feb 19;104(4):786-97. doi: 10.1016/j.bpj.2012.12.048. Gao X, Bai Y, Hwang TC
Cysteine scanning of CFTR's first transmembrane segment reveals its plausible roles in gating and permeation.
Biophys J. 2013 Feb 19;104(4):786-97. doi: 10.1016/j.bpj.2012.12.048., [PMID:23442957]

Abstract [show]
Previous cysteine scanning studies of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have identified several transmembrane segments (TMs), including TM1, 3, 6, 9, and 12, as structural components of the pore. Some of these TMs such as TM6 and 12 may also be involved in gating conformational changes. However, recent results on TM1 seem puzzling in that the observed reactive pattern was quite different from those seen with TM6 and 12. In addition, whether TM1 also plays a role in gating motions remains largely unknown. Here, we investigated CFTR's TM1 by applying methanethiosulfonate (MTS) reagents from both cytoplasmic and extracellular sides of the membrane. Our experiments identified four positive positions, E92, K95, Q98, and L102, when the negatively charged MTSES was applied from the cytoplasmic side. Intriguingly, these four residues reside in the extracellular half of TM1 in previously defined CFTR topology; we thus extended our scanning to residues located extracellularly to L102. We found that cysteines introduced into positions 106, 107, and 109 indeed react with extracellularly applied MTS probes, but not to intracellularly applied reagents. Interestingly, whole-cell A107C-CFTR currents were very sensitive to changes of bath pH as if the introduced cysteine assumes an altered pKa-like T338C in TM6. These findings lead us to propose a revised topology for CFTR's TM1 that spans at least from E92 to Y109. Additionally, side-dependent modifications of these positions indicate a narrow region (L102-I106) that prevents MTS reagents from penetrating the pore, a picture similar to what has been reported for TM6. Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR's gating cycle. The structural implications of these findings are discussed in light of the crystal structures of ABC transporters and homology models of CFTR.

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159 Although previous studies have provided evidence that these short-lived closures are ATP independent (43,44) and could result from voltage-dependent block of the pore by large anions from the cytoplasmic side of the channel (45), it is noted that these events are abundantly present in the double mutant L102C/E1371Q at both negative and positive membrane potentials in the absence of ATP (see Fig. S3). Login to comment
275 It is nevertheless interesting to note that in L102C/E1371Q-CFTR, the gate can open and close repeatedly even when the NBDs are in a dimeric configuration (Fig. S3). Login to comment

[hide] Relative contribution of different transmembrane s... Pflugers Arch. 2014 Mar;466(3):477-90. doi: 10.1007/s00424-013-1317-x. Epub 2013 Aug 20. Wang W, El Hiani Y, Rubaiy HN, Linsdell P
Relative contribution of different transmembrane segments to the CFTR chloride channel pore.
Pflugers Arch. 2014 Mar;466(3):477-90. doi: 10.1007/s00424-013-1317-x. Epub 2013 Aug 20., [PMID:23955087]

Abstract [show]
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) alpha-helices, arranged in 2 symmetrical groups of 6. However, those TMs that line the channel pore are not completely defined. We used patch clamp recording to compare the accessibility of cysteine-reactive reagents to cysteines introduced into different TMs. Several residues in TM11 were accessible to extracellular and/or intracellular cysteine reactive reagents; however, no reactive cysteines were identified in TMs 5 or 11. Two accessible residues in TM11 (T1115C and S1118C) were found to be more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels, as previously reported for T338C in TM6. However, the effects of mutagenesis at S1118 (TM11) on a range of pore functional properties were relatively minor compared to the large effects of mutagenesis at T338 (TM6). Our results suggest that the CFTR pore is lined by TM11 but not by TM5 or TM7. Comparison with previous works therefore suggests that the pore is lined by TMs 1, 6, 11, and 12, suggesting that the structure of the open channel pore is asymmetric in terms of the contributions of different TMs. Although TMs 6 and 11 appear to undergo similar conformational changes during channel opening and closing, the influence of these two TMs on the functional properties of the narrowest region of the pore is clearly unequal.

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46 In some cases, cysteine mutants were combined with the NBD2 mutation E1371Q, which we have used previously to abolish ATP-dependent channel gating and so isolate effects of cysteine reactive substances on open CFTR channels [41-43]. Login to comment
107 Figure 2a shows the effect of internal MTSES on currents carried by some TM11 mutant channels in inside- Fig. 4 Effect of the E1371Q mutation on modification by internal and external MTSES. Login to comment
111 b Example whole cell currents recorded continuously at +30 mV for constitutively active T1115C/ E1371Q and S1118C/E1371Q channels. Login to comment
115 In both c and d, black bars represent the named mutants in a cys-less background, and white bars in a cys-less/E1371Q background, and asterisks indicate a significant difference between these two backgrounds (P<0.05). Login to comment
136 This suggestion was based in large part on the effects of the E1371Q mutation, which results in constitutively open CFTR channels, on side-dependent modification. Login to comment
137 As shown in Fig. 4a, c, the E1371Q mutation significantly accelerated the rate of modification of both T1115C and S1118C by intracellular MTSES, suggesting that these cysteines are more readily modified from the inside in open channels. Login to comment
138 In contrast, these channels bearing the E1371Q mutation were almost completely insensitive to external MTSES (Fig. 4b), suggesting that these cysteines are much more difficult to modify from the outside in open channels (Fig. 4d). Login to comment

[hide] State-dependent blocker interactions with the CFTR... Pflugers Arch. 2014 Dec;466(12):2243-55. doi: 10.1007/s00424-014-1501-7. Epub 2014 Mar 28. Linsdell P
State-dependent blocker interactions with the CFTR chloride channel: implications for gating the pore.
Pflugers Arch. 2014 Dec;466(12):2243-55. doi: 10.1007/s00424-014-1501-7. Epub 2014 Mar 28., [PMID:24671572]

Abstract [show]
Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is subject to voltage-dependent open-channel block by a diverse range of cytoplasmic anions. However, in most cases the ability of these blocking substances to influence the pore opening and closing process has not been reported. In the present work, patch clamp recording was used to investigate the state-dependent block of CFTR by cytoplasmic Pt(NO2)4(2-) ions. Two major effects of Pt(NO2)4(2-) were identified. First, this anion caused fast, voltage-dependent block of open channels, leading to an apparent decrease in single-channel current amplitude. Secondly, Pt(NO2)4(2-) also decreased channel open probability due to an increase in interburst closed times. Interestingly, mutations in the pore that weakened (K95Q) or strengthened (I344K, V345K) interactions with Pt(NO2)4(2-) altered blocker effects both on Cl(-) permeation and on channel gating, suggesting that both these effects are a consequence of Pt(NO2)4(2-) interaction with a single site within the pore. Experiments at reduced extracellular Cl(-) concentration hinted that Pt(NO2)4(2-) may have a third effect, possibly increasing channel activity by interfering with channel closure. These results suggest that Pt(NO2)4(2-) can enter from the cytoplasm into the pore inner vestibule of both open and closed CFTR channels, and that Pt(NO2)4(2-) bound in the inner vestibule blocks Cl(-) permeation as well as interfering with channel opening and, perhaps, channel closure. Implications for the location of the channel gate in the pore, and the operation of this gate, are discussed.

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42 Some experiments were carried out with channels containing the NBD2 mutation E1371Q, which this laboratory [25-27, 29, 36] and others [2, 3] have used to abolish ATP-dependent channel gating and lock CFTR channels in the open state. Login to comment
43 Where the properties of different channel pore variants (wild type, K95Q, I344K, V345K) have been directly compared in wild type and E1371Q backgrounds, the wild-type background is referred to as "1371E" to indicate that the endogenous glutamate residue is present at this position. Login to comment
61 (F) Example leak-subtracted macroscopic I-V relationships for E1371Q-CFTR in the presence of 1 mM ATP, recorded before (control) and after the addition of 100 bc;M and 1 mM Pt(NO2)4 2-to the intracellular solution. Login to comment
62 (G, H) Mean data from experiments with E1371Q channels under the same conditions used for wild-type channels in (C-E). Login to comment
63 (I-K) Mean values of KD (at 0 mV in I and at +50 mV in J) and zb4; (K) obtained from fits of KD-Vrelationships such as those shown in (E) and (H), for both wild type and E1371Q channels under different nucleotide conditions as indicated. Login to comment
76 (C) Mean effect of this same concentration of Pt(NO2)4 2- on macroscopic current amplitudes under different conditions (black filled circle, wild type with 1 mM ATP; white empty circle, wild type with 1 mM ATP plus 2 mM PPi; filled down-pointing triangle E1371Q with 1 mM ATP), estimated from experiments such as those shown in Fig. 1. Login to comment
92 Block was also strongly voltage dependent when the channel open state was stabilized using the E1371Q mutation (Fig. 1(F-H)), which results in channel open probabilities close to one under these conditions [27]. Login to comment
93 Furthermore, in the E1371Q mutant, the blocking effects of Pt(NO2)4 2- were independent of either ATP concentration or the addition of 2 mM PPi (Fig. 1). Login to comment
95 Block of E1371Q was strongly voltage dependent under all conditions (Fig. 1(K)). Login to comment
98 The strongly voltage-dependent effects of this concentration of Pt(NO2)4 2- on unitary current amplitude (Fig. 2(B)) closely resemble the effects of this concentration of blocker on macroscopic wild-type currents following treatment with PPi, or in E1371Q mutant channels (Fig. 2(C, D)), whereas they are considerably weaker and more voltage-dependent than effects on macroscopic wild-type currents in the absence of PPi (Fig. 2(C, D)). Login to comment
106 When channel closure is inhibited using PPi or the E1371Q mutation, the effect on Cl-permeation is effectively isolated (Figs. 1 and 2). Login to comment
108 The effects of mutations in this region on the open-channel blocking effect of Pt(NO2)4 2- , isolated using E1371Q-containing channels, are shown in Fig. 3. Login to comment
110 Interestingly, not only blocker binding affinity (Fig. 3(D)) but also blocker voltage dependence (Fig. 3(E)) appeared correlated with the number of positively charged lysine side chains lining the inner vestibule of the pore; both I344K/E1371Q and V345K/E1371Q gave very strong, very strongly voltage-dependent block (Fig. 3(A, C-E)). Login to comment
111 To further investigate the relationship between Pt(NO2)4 2- block and gating, block of macroscopic currents was compared in 1371E (wild type) and E1371Q backgrounds (Fig. 4). Login to comment
112 The weak blocking effects of Pt(NO2)4 2- on K95Q-containing channels appeared independent of the presence of the E1371Q mutation (Fig. 4(B)), suggesting that no high-affinity Pt(NO2)4 2-binding site exists outside of the channel pore. Login to comment
113 In contrast, the strong inhibitory effects of Pt(NO2)4 2- on both I344K- and V345K-containing channels were observed in both E1371Q and 1371E backgrounds (Fig. 4(B)). Login to comment
114 Most strikingly, however, the very strong voltage dependence of block seen in both I344K/E1371Q and V345K/E1371Q (Fig. 3) was not observed in I344K/1371E and V345K/1371E channels that were allowed to open and close normally; instead, block was very strong but almost completely voltage-independent (Fig. 4). Login to comment
115 In many ways, these results with I344K and V345K recapitulate the results observed when comparing wild type and E1371Q channels (Fig. 1); block is weaker in E1371Q-containing channels (Fig. 4(C)), and blocker voltage dependence is greatly decreased (Fig. 4(D)). Login to comment
116 In fact, the discrepancy between 1371E and E1371Q channels, both in blocker affinity (Fig. 4(E)) and blocker voltage dependence (Fig. 4(F)), was greater when more fixed positive charges were present in the pore inner vestibule. Login to comment
123 Effect of extracellular Clon Pt(NO2)4 2- block As a consequence of the striking difference in voltage-dependence of block of I344K and V345K-containing channels studied in 1371E (Fig. 4) or E1371Q backgrounds (Fig. 3), there was an unusual "cross-over" effect in the relationship between the KD for Pt(NO2)4 2- block and voltage (Fig. 4(B)), suggesting that block is stronger in 1371E than in E1371Q channels at positive voltages, but weaker in 1371E at the most negative voltages studied. Login to comment
126 Under these conditions, cross-over of the KD-voltage relationship was observed for wild type and I344K channels, whereas for V345K channels block was now stronger in E1371Q channels at all voltages studied. Login to comment
128 (A) Example leak-subtracted macroscopic I-V relationships for the different E1371Q-containing CFTR constructs named, recorded before (control) and after the addition of Pt(NO2)4 2-to the intracellular solution at the concentrations indicated. Login to comment
131 (D, E) Mean values of KD (at 0 mV) and zb4; obtained from fits of KD-V relationships such as those shown in (C), as a function of the number of positively charged lysine side chains lining the inner vestibule of the pore in different channel constructs. Asterisks indicate a significant difference from wild type (E1371Q) (P<0.005). Login to comment
133 However, whereas the Pt(NO2)4 2- -induced decrease in PO could easily be studied at the single-channel level at depolarized voltages at which open-channel blocking effects are practically absent (Figs. 2 and 5), the discrepancy between the KD measured in 1371E and E1371Q channels shown in Fig. 6(C) was most striking at hyperpolarized voltages at which effects on single-channel current amplitudes (as reflected by effects on E1371Q channels) are expected to be very strong, and could not therefore be investigated directly using single-channel recordings. Login to comment
135 Under these ionic conditions, the E1371Q mutation increased the apparent affinity of block (Fig. 7(A, B)), an effect that was especially strong in I344K and V345K channels and at hyperpolarized voltages. Login to comment
136 This contrasts with what was found under high [Cl- ] conditions, where the E1371Q mutation decreased the affinity of block, especially in I344K and V345K (Fig. 4(C)). Login to comment
137 The E1371Q mutation also increased the voltage dependence of block under low [Cl- ] Fig. 4 Gating-dependent Pt(NO2)4 2- block in pore mutant forms of CFTR. Login to comment
139 (B) Mean KD values obtained from such data as described in Figs. 1 and 3 (white empty circle), compared to data from the same channel mutants in an E1371Q background (black filled circle) (taken from Fig. 3(C)). Login to comment
140 (C, D) Comparison of mean KD (at 0 mV) and zb4; values estimated for different channel variants in an E1371Q background (black bars) versus a 1371E background (grey bars). Login to comment
141 Asterisks indicate a significant difference from the same channel variant in an E1371Q background (P<0.0005). Login to comment
142 (E, F) Direct quantification of the effect of different E1371 background on KD (at 0 mV) and zb4;, evaluated as the mean value in a 1371E background as a fraction of that in an E1371Q background, and plotted relative to the number of positively charged lysine side chains lining the inner vestibule of the pore in different channel constructs. Asterisks indicate a significant difference from the value estimated for wild type (P<0.05). Login to comment
146 However, [Cl- ] has little effect on the voltage dependence of block (Fig. 7(E)), which is already very strong in I344K/E1371Q and V345K/E1371Q channels (Figs. 4(D) and 7(C)). Login to comment
159 Multiple effects of Pt(NO2)4 2- on conductance and gating The open-channel blocking effects of Pt(NO2)4 2are the easiest to isolate and observe-these effects can be seen from the effect of Pt(NO2)4 2- on single-channel current amplitude (Fig. 2(A, B)) and are also isolated in channels in which the open state has been stabilized, either using PPi to interrupt normal ATP-dependent gating (Fig. 1) or using the ATP hydrolysis-defective E1371Q mutant (Figs. 1, 3, and 6). Login to comment
162 (A, B) Example leak-subtracted macroscopic I-V relationships for the different CFTR constructs named, either in an E1371Q background (A) or a 1371E background (B), with a low (4 mM) [Cl- ] extracellular solution. Login to comment
176 In each of these situations, block at very negative voltages appears anomalously weak in 1371E channels when compared to E1371Q, hinting that Pt(NO2)4 2- block might actually be associated with an increase in PO under these conditions. Login to comment
181 In each panel, black bars represent an E1371Q background and grey bars a 1371E background; asterisks indicate a significant difference from the same channel variant in an E1371Q background (P<0.05); and (in D and E) daggers represent a significant difference between high and low external [Cl- ] conditions (P<0.05). Login to comment
193 Effects on open channels are isolated in E1371Q channels. Login to comment
195 Inhibitory effects on PO are due to effects on closed channels (increasing interburst duration), and so are most prominent in macroscopic current recordings under conditions in which open-channel block is relatively weak (e.g., at positive voltages), leading to the largest discrepancy in overall blocking effects between 1371E and E1371Q channels (Figs. 1, 2(C), and 4(B)). Login to comment
198 Effect of extracellular Cl- Increasing extracellular [Cl- ] weakens Pt(NO2)4 2- block of open E1371Q channels (Fig. 7(D)), as described previously for wild-type CFTR [11, 35]. Login to comment
200 Interestingly, this apparent knock-off effect is greatly strengthened in I344K/E1371Q and V345K/E1371Q (Fig. 7(D)), suggesting Fig. 8 Schematic summary of the effects of Pt(NO2)4 2- . Login to comment
203 Experiments carried out on E1371Q channels are assumed to isolate effects on open channels (to the right of the dotted line in (A)). Login to comment
206 Block of macroscopic 1371E channels is generally stronger than that of E1371Q channels at high extracellular [Cl- ] (Fig. 1(E) and 4(B)), whereas block is generally weaker in 1371E channels at low extracellular [Cl- ] (Fig. 6(C)). Login to comment

[hide] Metal bridges illuminate transmembrane domain move... J Biol Chem. 2014 Oct 10;289(41):28149-59. doi: 10.1074/jbc.M114.593103. Epub 2014 Aug 20. El Hiani Y, Linsdell P
Metal bridges illuminate transmembrane domain movements during gating of the cystic fibrosis transmembrane conductance regulator chloride channel.
J Biol Chem. 2014 Oct 10;289(41):28149-59. doi: 10.1074/jbc.M114.593103. Epub 2014 Aug 20., [PMID:25143385]

Abstract [show]
Opening and closing of the cystic fibrosis transmembrane conductance regulator are controlled by ATP binding and hydrolysis by the cytoplasmic nucleotide-binding domains. Different conformational changes in the channel pore have been described during channel opening and closing; however, the relative importance of these changes to the process of gating the pore is not known. We have used patch clamp recording to identify high affinity Cd(2+) bridges formed between pairs of pore-lining cysteine residues introduced into different transmembrane alpha-helices (TMs). Seven Cd(2+) bridges were identified forming between cysteines in TMs 6 and 12. Interestingly, each of these Cd(2+) bridges apparently formed only in closed channels, and their formation stabilized the closed state. In contrast, a single Cd(2+) bridge identified between cysteines in TMs 1 and 12 stabilized the channel open state. Analysis of the pattern of Cd(2+) bridge formation in different channel states suggests that lateral separation and convergence of different TMs, rather than relative rotation or translation of different TMs, is the key conformational change that causes the channel pore to open and close.

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49 In some cases, cysteine mutants were combined with the NBD2 mutation E1371Q, which we (7-9) and others (12, 26) have used to abolish ATP-dependent channel gating and lock CFTR channels in the open state. Login to comment
50 In our expression system, the open probability of channels bearing the E1371Q mutation is b0e;95% (7). Login to comment
82 To confirm this apparent state dependence of Cd2af9; bridge formation, we combined selected single and double cysteine mutants with the E1371Q mutation in NBD2 that results in constitutively open channels in our expression system (see "Experimental Procedures"). Login to comment
83 As shown in Fig. 5, all E1371Q-containing channels tested were only weakly sensitive to inhibition by Cd2af9; , resulting in a significant increase in Ki both in single cysteine (I344C, M348C, S1141C) and in double cysteine (I344C/S1141C, Fig. 5, A-C; M348C/S1141C, Fig. 5, A, D, and E) mutant channels. Login to comment
84 However, the effect of the E1371Q mutation was greater in the double cysteine mutants; this gating mutation increased Ki 30-fold in I344C/S1141C (Fig. 5C) and 2500-fold in M348C/S1141C (Fig. 5E). Login to comment
90 Conformational Changes during CFTR Channel Opening OCTOBER 10, 2014ߦVOLUME 289ߦNUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 28151 ing single cysteine mutants (Figs. 3 and Fig. 5, C and E), in E1371Q mutant channels, the Ki for the double cysteine mutants was not significantly different from the single cysteine mutants (Fig. 5, B-E). Login to comment
109 However, we found that constitutively open K95C/E1371Q channels were insensitive to Cd2af9; at concentrations up to 100 òe;M (Fig. 6B), as were K95C channels following treatment with 2 mM PPi (data not shown), inconsistent with an effect of Cd2af9; on Clafa; conductance. Login to comment
110 Instead, these results suggest that Cd2af9; binding to K95C results in a small but significant stabilization of the channel open state, an effect that is not observed in E1371Q channels that are already open almost 100% of the time (7). Login to comment
116 As with K95C itself, the stimulatory effects of Cd2af9; on K95C/S1141C were abolished by the E1371Q mutation (Fig. 6C) or by treatment with PPi (data not shown), suggesting that the observed effects of Cd2af9; are due to an increase in channel open probability and a stabilization of the channel open state. Login to comment
145 Thus, if channels are stabilized in the open state, either using PPi to manipulate NBD-driven gating (Fig. 4) or using E1371Q channels that cannot hydrolyze ATP (Fig. 5), the inhibitory effects of Cd2af9; are greatly reduced, suggesting that Cd2af9; binds more strongly to the closed state than to the open state. Login to comment
151 Error bars indicate the means afe; S.E. from 3-7 patches. Conformational Changes during CFTR Channel Opening 28154 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289ߦNUMBER 41ߦOCTOBER 10, 2014 at SEMMELWEIS UNIV OF MEDICINE on December 4, that appears to bind Cd2af9; b;2500 times more strongly in channels that are gating normally as compared with constitutively open E1371Q channels (Fig. 5). Login to comment
153 However, when the channel is open, these two cysteines do not appear to coordinate Cd2af9; at all because the apparent Cd2af9; affinity in M348C/S1141C/ E1371Q channels appears the same as in M348C/E1371Q or S1141C/E1371Q (Fig. 5, D and E). Login to comment
155 Because this effect was abolished in E1371Q channels (Fig. 6), it appears to reflect a Cd2af9; bridge-induced change in channel gating rather than channel conductance. Login to comment
163 Effect of the E1371Q mutation on the effects of Cd2d19; on single and double cysteine mutant channels in TMs 6 and 12. Login to comment
164 A, sample time courses and I-V curves illustrating the low Cd2af9; sensitivity of constitutively active I344C/S1141C/E1371Q (left panels) and M348C/S1141C/E1371Q (right panels) channels in inside-out patches. Experiments were performed as described in the legend for Fig. 2. Login to comment
166 B-E, mean fractional current remaining following the addition of differentconcentrationsofCd2af9; (BandD)andmeanKi valuesforthesingleanddoublecysteinemutantslisted(CandE).InCandE,blackbarsindicatechannels gating normally, and gray bars indicate the corresponding cysteine mutants with the E1371Q mutation; asterisks indicate a significant difference between the two for the same cysteine constructs (p b0d; 0.02). Login to comment
179 B, concentration-dependent increases in current amplitude in K95C were not observed in K95C/E1371Q. Login to comment
181 C, Cd2af9; causes a significantly greater increase in K95C/S1141C current amplitude as compared with K95C (p b0d; 0.0001); this increase is not observed in K95C/S1141C/E1371Q channels. Login to comment

[hide] The cystic fibrosis transmembrane conductance regu... Pflugers Arch. 2015 Aug;467(8):1783-94. doi: 10.1007/s00424-014-1618-8. Epub 2014 Oct 4. Broadbent SD, Ramjeesingh M, Bear CE, Argent BE, Linsdell P, Gray MA
The cystic fibrosis transmembrane conductance regulator is an extracellular chloride sensor.
Pflugers Arch. 2015 Aug;467(8):1783-94. doi: 10.1007/s00424-014-1618-8. Epub 2014 Oct 4., [PMID:25277268]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel that governs the quantity and composition of epithelial secretions. CFTR function is normally tightly controlled as dysregulation can lead to life-threatening diseases such as secretory diarrhoea and cystic fibrosis. CFTR activity is regulated by phosphorylation of its cytosolic regulatory (R) domain, and ATP binding and hydrolysis at two nucleotide-binding domains (NBDs). Here, we report that CFTR activity is also controlled by extracellular Cl(-) concentration ([Cl(-)]o). Patch clamp current recordings show that a rise in [Cl(-)]o stimulates CFTR channel activity, an effect conferred by a single arginine residue, R899, in extracellular loop 4 of the protein. Using NBD mutants and ATP dose response studies in WT channels, we determined that [Cl(-)]o sensing was linked to changes in ATP binding energy at NBD1, which likely impacts NBD dimer stability. Biochemical measurements showed that increasing [Cl(-)]o decreased the intrinsic ATPase activity of CFTR mainly through a reduction in maximal ATP turnover. Our studies indicate that sensing [Cl(-)]o is a novel mechanism for regulating CFTR activity and suggest that the luminal ionic environment is an important physiological arbiter of CFTR function, which has significant implications for salt and fluid homeostasis in epithelial tissues.

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92 We then examined whether the ATP hydrolysis-deficient CFTR mutant, E1371Q [38], could sense [Cl- ]o with the expectation that it would not. Login to comment
93 Like WT CFTR exposed to AMP-PNP, the E1371Q mutant channels display extended open channel bursts in the presence of ATP [16, 23, 40]. Login to comment
94 In our experiments, E1371Q CFTR expressed in HEK cells was constitutively active and large currents were detected without prior stimulation with FSK (Fig. 3d (i) (first set of fWCR current traces), and 3e), which agrees with previous work performed in baby hamster kidney (BHK) cells [27, 42, 46]. Login to comment
95 The reason for this constitutive activity of E1371Q is unclear and it contrasts with the behaviour of wild type CFTR and other mutants (except DeltaR-CFTR) previously investigated in our laboratories using the BHK and HEK cell expression systems [46]. Login to comment
96 Exposing E1371Q CFTR (without FSK) to an increase in [Cl- ]o caused only a small stimulation of channel activity (21.9&#b1;14.1 %, n=6; Fig. 3f). Login to comment
97 Surprisingly in view of the WT CFTR data with AMP-PNP, if E1371Q CFTR channels were first phosphorylated with FSK (which itself had no significant effect on the size of the whole cell currents (Fig. 3d (ii) (second set of fWCR traces)) and Table 1), [Cl- ]o sensing was restored (174.5&#b1;56.2 % stimulation, n=8; Fig. 3d, e and f). Login to comment
98 To confirm the role of phosphorylation in [Cl- ]o sensing, we reduced the endogenous phosphorylation of E1371Q channels by removing ATP/GTP from the intracellular solution prior to stimulation with FSK, which significantly reduced basal E1371Q CFTR currents (No ATP/GTP: -186&#b1; 122 pA/pF n=8 vs ATP/GTP: -1,259&#b1;433 pA/pF, n=8, P<0.001). Login to comment
99 Under these conditions, the E1371Q CFTR channels failed to respond to changes in [Cl- ]o even in the presence of FSK (Fig. 3f), consistent with the idea that only phosphorylated channels are gated by [Cl- ]o. Login to comment
100 Since E1371Q CFTR is hydrolysis-deficient [38], we expected that [Cl- ]o sensing by the mutant would not be affected by AMP-PNP. Login to comment
101 However, unexpectedly, we found that the ability of FSK-stimulated E1371Q CFTR channels to respond to changes in [Cl- ]o was markedly reduced by the non-hydrolysable ATP analogue, just like WT CFTR (Fig. 3f). Login to comment
102 To check that the restoration of [Cl- ]o sensing by E1371Q CFTR after phosphorylation (Fig. 3d) was due to an C A B WT R899Q VEC WT (i) (ii) (iii) (iv) (i) (ii) (iii) (iv) R899Q Fig. 2 Arginine residue 889 in extracellular loop 4 of CFTR is essential for [Cl- ]o sensing. Login to comment
110 **p<0.01 compared to WT CFTR interaction of Cl-with the extracellular domain of CFTR, we also studied the double-mutant R899Q-E1371Q. Login to comment
111 Figure 3f shows that [Cl- ]o sensing by the double mutant in the presence of FSK was significantly reduced compared to E1371Q CFTR under the same conditions, confirming that phosphorylated E1371Q CFTR channels respond to changes in [Cl- ]o via R899 in ECL4. Login to comment
112 To explore the role of phosphorylation further, we studied the effect of deleting the R domain from CFTR (residues 634-836) [12, 7], which removes all the major PKA/PKC Table 1 Summary of the FSK stimulation of whole cell currents and Erev shifts observed with the CFTR constructs used in this study CFTR Construct n FSK Stimulation (%&#b1;SEM) Erev shift (mV&#b1;SEM) WT (50 bc;M ATP) 5 180&#b1;96 15.0&#b1;3.6 WT (100 bc;M ATP) 6 12,000&#b1;6,000 15.2&#b1;3.0 WT (300 bc;M ATP) 8 1,200&#b1;600 17.0&#b1;3.0 WT (1 mM ATP) 24 13,000&#b1;6,000 23.7&#b1;1.8 WT (1.3 mM ATP) 9 1,400&#b1;900 16.7&#b1;2.6 WT (2 mM ATP) 24 6,100&#b1;5,300 16.7&#b1;1.6 WT (5 mM ATP) 7 1,600&#b1;1,000 20.1&#b1;4.4 WT (50 bc;M ATP + 50 bc;M P-ATP) 7 224&#b1;130 15.3&#b1;1.0 WT + Genistein 4 7,600&#b1;5,200 26.1&#b1;5.4 WT + AMP-PNP 5 2,800&#b1;2,500 21.8&#b1;5.5 WT (3 mM MgCl2) 7 28,000&#b1;17,000 18.3&#b1;3.1 R104Q 5 4,600&#b1;1,600 28.6&#b1;4.7 K114C 5 12,000&#b1;6,700 29.2&#b1;3.0 R117Q 4 33,000&#b1;20,000 30.1&#b1;3.4 K329A 5 13,000&#b1;10,000 33.7&#b1;2.1 R334Q 9 13,000&#b1;6,700 27.3&#b1;2.9 K335A 5 3,200&#b1;1,500 20.8&#b1;7.1 W401G 7 2,600&#b1;1,800 18.5&#b1;4.8 Delta-R (No Stim) 5 - 25.1&#b1;2.7 Delta-R (No FSK, Genistein) 5 140&#b1;13 22.7&#b1;3.0 Delta-R (FSK, No Genistein) 4 89&#b1;14 15.6&#b1;6.0 Delta-R (FSK + Genistein) 6 639&#b1;432 25.1&#b1;4.9 Delta-R-E1371S (No FSK) 9 - 21.4&#b1;4.8 Delta-R-E1371S (FSK) 4 2,600&#b1;1,400 15.3&#b1;4.7 K892Q 7 16,000&#b1;9,500 36.8&#b1;4.8 R899E 4 1,200&#b1;400 25.0&#b1;2.7 R899K 4 1,600&#b1;900 26.6&#b1;2.9 R899Q 7 5,400&#b1;2,800 30.0&#b1;1.3 R899Q + AMP-PNP 4 72,000&#b1;50,000 15.2&#b1;2.8 R899Q-E1371Q (No FSK) 4 - 18.4&#b1;5.9 R899Q-E1371Q (FSK) 6 107&#b1;48 15.6&#b1;3.0 R1128Q 6 14,000&#b1;6,100 41.1&#b1;4.2 Y1219G 6 3,200&#b1;2,500 19.2&#b1;3.3 E1371Q (No FSK) 6 - 25.5&#b1;3.5 E1371Q (FSK) 8 -28&#b1;9 22.3&#b1;4.0 E1371Q (FSK, No ATP, No GTP) 8 270&#b1;130 19.4&#b1;4.5 E1371Q + AMP-PNP (No FSK) 4 - 24.7&#b1;6.5 E1371Q + AMP-PNP (FSK) 8 180&#b1;170 17.4&#b1;4.0 Vector Control 4 15&#b1;38 - FSK stimulation was calculated as the percentage increase in current density at -60 mV from the Erev, after 5-min exposure to 10 bc;M FSK. Login to comment
120 Based on our previous result that the phosphorylated, hydrolysis-deficient, E1371Q mutant could sense [Cl- ]o (Fig. 3f), we predicted that preventing ATP hydrolysis at ATP binding site 2 of DeltaR-CFTR would have no effect on [Cl- ]o sensing. Login to comment
121 We tested this prediction by using DeltaR-CFTR with the E1371S mutation, which like E1371Q CFTR is hydrolysis defective [8] and found, unexpectedly, that the double-mutant CFTR channels did not respond to changes in [Cl- ]o (external Cl- stimulation; no FSK/genistein: 0.0&#b1;7.0 %, n=9; with FSK/genistein: 16.7&#b1; 7.7 %, n=4) (Fig. 4c-e). Login to comment
122 Role of ATP binding and ATPase activity of the NBDs in [Cl- ]o sensing by CFTR Figures 3 and 4 suggest that the role of ATP hydrolysis in [Cl- ]o sensing by CFTR is complex; e.g. AMP-PNP abolishes WT + AMP-PNP 4 nA 100 ms (i) (ii) (iii) (iv) E1371Q (FSK) 100 ms 30 nA (i) (ii) (iii) (iv) E1371Q (FSK) WT + AMP-PNP A E D C B F Fig. 3 Gating of CFTR by [Cl- ]o requires ATP hydrolysis and phosphorylation. Login to comment
125 b, d Representative fWCR current recordings measured between &#b1;100 mV in 20 mV steps from HEK cells transfected with WT CFTR plus AMP-PNP and E1371Q CFTR, as indicated. The current traces are from the top down: (i) unstimulated in 155.5 mM [Cl- ]o, (ii) forskolin (FSK)-stimulated in 155.5 mM [Cl- ]o, (iii) FSK-stimulated in 35.5 mM [Cl- ]o and (iv) FSK-stimulated in 155.5 mM [Cl- ]o. Dotted line to the right of the current traces indicates zero current level. Login to comment
126 c, e Representative I-V plots for the data presented in b and d. f Percentage stimulation of either basal or FSK-activated currents by [Cl- ]o for WT CFTR (n=24), the ECL4 mutant R899Q, the hydrolysis-deficient mutant E1371Q and the double-mutant R899Q-E1371Q (see Fig. 1) under different conditions as indicated (n=4-8). Login to comment
128 *p<0.05, **p<0.01, ***p<0.001 between indicated datasets [Cl- ]o sensing by WT CFTR, whereas the E1371Q mutation has no effect. Login to comment
143 The current traces are from the top down: (i) unstimulated in 155.5 mM [Cl- ]o, (ii) unstimulated in 35.5 mM [Cl- ]o and (iii) unstimulated in 155.5 mM [Cl- ]o. Dotted line to the right of the current traces indicates zero current level. b, d Representative I-V plots for the data presented in a and c. e Percentage current stimulation by [Cl- ]o for WT CFTR (n=24) and for DeltaR and DeltaR-E1371Q mutants (see Fig. 1) under unstimulated (No Stim) or after exposure to a combination of forskolin and genistein (FSK/Genistein). Login to comment
166 First, cAMP/PKA-dependent phosphorylation is required for the response in WTand E1371Q CFTR channels. Login to comment
172 Secondly, in phosphorylated CFTR channels, impairing ATP hydrolysis using the E1371Q mutation did not prevent [Cl- ]o sensing. This result implies that chloride sensing does not lead to a change in channel gating simply via a change in ATPase activity. Login to comment
174 The fact that [Cl- ]o sensing is impaired in phosphorylated E1371Q CFTR channels when AMP-PNP is present, is probably explained by AMP-PNP substituting for ATP in its actions. Login to comment
175 As Fig. 5f shows, raising cytosolic [ATP] to 2 mM, or above, abolished sensing. This is why AMP-PNP effects are mimicked by ATP, but not by the E1371Q mutation. Login to comment
176 In this context, it is important to remember that although the E1371Q mutation substantially reduces ATP hydrolysis (~70 %, [38]), the channel is still gated by ATP, even in a DeltaR background [12, 7, 8]. Login to comment

[hide] Location of a permeant anion binding site in the c... J Physiol Sci. 2015 May;65(3):233-41. doi: 10.1007/s12576-015-0359-6. Epub 2015 Feb 12. Rubaiy HN, Linsdell P
Location of a permeant anion binding site in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Physiol Sci. 2015 May;65(3):233-41. doi: 10.1007/s12576-015-0359-6. Epub 2015 Feb 12., [PMID:25673337]

Abstract [show]
In the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, lyotropic anions with high permeability also bind relatively tightly within the pore. However, the location of permeant anion binding sites, as well as their relationship to anion permeability, is not known. We have identified lysine residue K95 as a key determinant of permeant anion binding in the CFTR pore. Lyotropic anion binding affinity is related to the number of positively charged amino acids located in the inner vestibule of the pore. However, mutations that change the number of positive charges in this pore region have minimal effects on anion permeability. In contrast, a mutation at the narrow pore region alters permeability with minimal effects on anion binding. Our results suggest that a localized permeant anion binding site exists in the pore; however, anion binding to this site has little influence over anion permeability. Implications of this work for the mechanisms of anion recognition and permeability in CFTR are discussed.

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41 In contrast, the F337A mutation disrupts the normal Fig. 1 Block by intracellular Au(CN)2 - is weakened in K95Q/E1371Q channels. Example macroscopic IV relationships for E1371Q (a) and K95Q/E1371Q (b) CFTR channels recorded before (control) and after the addition of Au(CN)2 - to the intracellular (bath) solution at the concentrations stated. Login to comment
48 In order to isolate anion effects on Cl-permeation from effects on channel gating [17, 24], experiments were carried out on constitutively active E1371Q-CFTR channels [22, 24]. Login to comment
49 Additional mutations were introduced into this E1371Q background using the QuikChange site-directed mutagenesis system (Agilent Technologies, Santa Clara, CA, USA) and verified by DNA sequencing. Login to comment
50 Macroscopic E1371Q-CFTR current-voltage (I-V) relationships were recorded using depolarizing voltage ramps during patch clamp recordings from inside-out membrane patches, as described in detail recently [24]. Login to comment
57 The macroscopic current reversal potential (VREV) Fig. 2 Block by intracellular SCN- and C(CN)3 - is weakened in K95Q/E1371Q channels. Example macroscopic IV relationships for E1371Q (a) and K95Q/E1371Q (b) CFTR channels recorded before (control) and after the addition of 10 mM SCN- to the intracellular (bath) solution. c Mean KD values for SCN- block for these channel constructs, obtained as described for Au(CN)2 - in Fig. 1. Login to comment
68 Results Intracellular Au(CN)2 - ions cause a voltage-dependent block of open CFTR channel pores [17], a finding that is recapitulated in constitutively open E1371Q channels (Fig. 1). Login to comment
71 Thus, the mean KD was increased between 92-fold (at -100 mV) and 19-fold (at ?60 mV) in K95Q/E1371Q (Fig. 1d). Login to comment
72 Similar weakening of block in K95Q/E1371Q Fig. 3 Strength of Au(CN)2 - block is dependent on the number of positive charges in the pore inner vestibule. Login to comment
73 a Example macroscopic I-V relationships for K95H/E1371Q CFTR channels recorded using bath solutions at pH 5.5 (left) or pH 9.0 (right, different patch). Login to comment
75 b Mean KD values for Au(CN)2 - block of K95H/E1371Q at these two different pHs, obtained as described for Au(CN)2 - in Fig. 1. Login to comment
77 c Relationship between the observed KD values for Au(CN)2 - block (at -100 mV) and bath solution pH in K95H/ E1371Q and E1371Q. Login to comment
78 d Example macroscopic I-V relationships for I344K/E1371Q (left) and K95Q/I344K/E1371Q (right) CFTR channels recorded before (control) and after the addition of a low concentration (10 lM) of Au(CN)2 - to the intracellular (bath) solution. Login to comment
79 e Mean KD values for Au(CN)2 - block for these channel constructs, as well as the additional positive charge mutants V345K/ E1371Q and S1141K/E1371Q, obtained as described in Fig. 1. f Relationship between the observed KD values for Au(CN)2 - block (at -100 mV) and the expected number of fixed positive charges in the pore inner vestibule in different channel constructs. Login to comment
82 Asterisks indicate a significant difference from E1371Q (*P \ 0.05, **P \ 0.0001). Login to comment
86 As shown in Fig. 3a and b, block of K95H/E1371Q by intracellular Au(CN)2 - was drastically stronger at pH 5.5 compared to pH 9.0, with the mean KD at -100 mV being increased 244-fold at the more alkaline pH. Login to comment
87 In contrast, Au(CN)2 - block of E1371Q was not pH-dependent (Fig. 3c). Login to comment
90 Consistent with this, mutation of any of these three residues to lysine (I344K, V345K, S1141K) led to a significant strengthening of Au(CN)2 - block (Fig. 3d-f), with mean KD values at -100 mV being reduced by 18-fold (I344K/E1371Q), 17-fold (V345K/E1371Q) and 7-fold (S1141K/E1371Q), respectively. Login to comment
91 ''Moving`` the key positive charge from TM1 to TM6 by mutagenesis (in the K95Q/I344K/E1371Q mutant) resulted in Au(CN)2 - block that was intermediate in strength between E1371Q and I344K/E1371Q (Fig. 3e-f). Login to comment
93 As well as showing tight binding of lyotropic permeant anions, CFTR also shows a lyotropic anion permeability Fig. 4 Block of F337A/ E1371Q channels by intracellular lyotropic permeant anions. Login to comment
94 a, b Example macroscopic I-V relationships for F337A/E1371Q CFTR channels recorded before (control) and after the addition of Au(CN)2 - (1 mM) or SCN- (10 mM) to the intracellular (bath) solution. c Mean KD values for Au(CN)2 - , SCN- , and C(CN)3 - (estimated at -100 mV as described in Figs. 1 and 2) compared in E1371Q, K95Q/ E1371Q, and F337A/E1371Q. Login to comment
95 Asterisks indicate a significant difference from E1371Q (P \ 0.01). Login to comment
100 As shown in Fig. 5, macroscopic current reversal potential measurements with different anions present in the intracellular solution gave a permeability selectivity sequence SCN- [ NO3 - [ Br- [ Cl- [ F- for E1371Q-CFTR, with very similar relative permeabilities for those previously reported for wild type CFTR under similar conditions [11]. Login to comment
101 Consistent with the proposed role of F337 in controlling lyotropic anion permeability, the permeability of all anions tested in F337A/E1371Q was significantly changed relative to E1371Q (Fig. 5b), with the permeability selectivity sequence being changed to NO3 - - C SCN- C Cl- [ Br- [ F- . Login to comment
102 Again, relative permeability values for F337A/E1371Q were similar to those reported previously for F337A [18]. Login to comment
103 In contrast, neither K95Q nor I344K altered the permeability selectivity sequence; in fact, the only significant difference between either K95Q/ E1371Q or I344K/E1371Q compared to E1371Q was a small increase in PF/PCl in K95Q/E1371Q. Login to comment
106 Note that the range of current reversal potentials was greatly reduced in F337A/E1371Q, suggesting a relative loss of permeability selectivity in this mutant. Login to comment
107 b Mean PX/PCl values calculated from current reversal potential measurements under these conditions as described in ''Materials and methods.`` Asterisks indicate a significant difference from E1371Q (*P \ 0.05; **P \ 0.002). Login to comment
109 Note that the normal lyotropic relationship between relative permeability and Gh is greatly reduced in F337A/E1371Q but retained in K95Q/E1371Q and I344K/E1371Q. Login to comment

[hide] Interactions between permeant and blocking anions ... Biochim Biophys Acta. 2015 Jul;1848(7):1573-90. doi: 10.1016/j.bbamem.2015.04.004. Epub 2015 Apr 17. Linsdell P
Interactions between permeant and blocking anions inside the CFTR chloride channel pore.
Biochim Biophys Acta. 2015 Jul;1848(7):1573-90. doi: 10.1016/j.bbamem.2015.04.004. Epub 2015 Apr 17., [PMID:25892339]

Abstract [show]
Binding of cytoplasmic anionic open channel blockers within the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is antagonized by extracellular Cl(-). In the present work, patch clamp recording was used to investigate the interaction between extracellular Cl(-) (and other anions) and cytoplasmic Pt(NO2)4(2-) ions inside the CFTR channel pore. In constitutively open (E1371Q-CFTR) channels, these different anions bind to two separate sites, located in the outer and inner vestibules of the pore respectively, in a mutually antagonistic fashion. A mutation in the inner vestibule (I344K) that greatly increased Pt(NO2)4(2-) binding affinity also greatly strengthened antagonistic Cl(-):blocker interactions as well as the voltage-dependence of block. Quantitative analysis of ion binding affinity suggested that the I344K mutation strengthened interactions not only with intracellular Pt(NO2)4(2-) ions but also with extracellular Cl(-), and that altered blocker Cl(-)- and voltage-dependence were due to the introduction of a novel type of antagonistic ion:ion interaction inside the pore that was independent of Cl(-) binding in the outer vestibule. It is proposed that this mutation alters the arrangement of anion binding sites inside the pore, allowing both Cl(-) and Pt(NO2)4(2-) to bind concurrently within the inner vestibule in a strongly mutually antagonistic fashion. However, the I344K mutation does not increase single channel conductance following disruption of Cl(-) binding in the outer vestibule in R334Q channels. Implications for the arrangement of ion binding sites in the pore, and their functional consequences for blocker binding and for rapid Cl(-) permeation, are discussed.

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2 In constitutively open (E1371Q-CFTR) channels, these different anions bind to two separate sites, located in the outer and inner vestibules of the pore respectively, in a mutually antagonistic fashion. Login to comment
32 Binding of Pt(NO2)4 2-to this site in the inner vestibule affects both Cl-permeation through the open channel and also channel opening and closing [47], however the effect on Cl- movement in open channels can be isolated using gating-defective, constitutively open E1371Q-CFTR channels [47]. Login to comment
40 Macroscopic currents were recorded from gating-defective, constitutively-open E1371Q channels [49], whereas single channel recording experiments were carried out in a wild type CFTR background. Login to comment
41 It has previously been shown that, whereas Pt(NO2)4 2- has complex effects on Cl- conduction and gating in wild type, effects on conduction are isolated in E1371Q, and that blocking effects on this channel mutant are indistinguishable from those on wild type channels in the open state [47]. Login to comment
65 (A, B, E, F) Example macroscopic I-V relationships for E1371Q (A, B) or R899Q/E1371Q (E, F) under high (154 mM; A, E) or low (4 mM; B, F) extracellular [Cl- ] conditions. In each case currents were recorded before (control) and after the addition of 100 bc;M and 1 mM Pt(NO2)4 2-to the intracellular solution. Login to comment
66 (C, G) Mean fraction of control current remaining after addition of different concentrations of Pt(NO2)4 2- at a membrane potential of -100 mV for E1371Q (C) and R899Q/E1371Q (G) under high (filled circles) and low (open circles) extracellular [Cl- ] conditions. Data have been fitted using Eq. (1) as described in the Materials and methods. Login to comment
67 (D) Mean Pt KD values obtained from such fits at different membrane potentials for E1371Q; results for R899Q/E1371Q were indistinguishable (not shown). Login to comment
69 (H) Mean values of Pt KD at 0 mV membrane potential obtained from fits of Pt KD-V relationships such as those shown in D for E1371Q (filled squares) and R899Q/E1371Q (open squares) at different extracellular [Cl- ]. Login to comment
71 There was no significant difference between E1371Q and R899Q/E1371Q (P N 0.85). Login to comment
73 dissociation constant for block by intracellular Pt(NO2)4 2- ions (Pt KD) at different extracellular [Cl- ] ([Cl- ]o) derives from both the dissociation constant for the internal binding site when the external binding site is occupied by Cl- (Pt Kocc) and that when the external binding site is vacant (Pt Kvac) according to: Pt KD &#bc; Pt Kocc Cl - &#bd; o &#fe; Pt Kvac: &#f0;3&#de; This model predicts a linear relationship between Pt KD and [Cl- ]o, which was reasonably well observed in the present study at different membrane potentials for E1371Q (Figs. 2H, 8A) and pore-mutant CFTR channels (Figs. 4E, J, 6D, H, 8B, C, 10A, B), as well as when extracellular Cl-was replaced by other anions (Figs. 3D, 7E, F, 11A, E). Login to comment
82 This effect is illustrated for constitutively open E1371Q channels in Fig. 2: Pt(NO2)4 2- block is strengthened as extracellular Cl- is replaced by impermeant gluconate anions (Fig. 2C, D), resulting in a monotonic increase in the measured KD (at 0 mV membrane potential) as extracellular [Cl- ] is increased (Fig. 2H). Login to comment
83 Identical results were obtained in R899Q/ E1371Q channels (Fig. 2; Table 1), both at high and low [Cl- ]o conditions, indicating that extracellular Cl- interactions with this externally-located arginine residue (Fig. 1) do not contribute to these antagonistic effects. Login to comment
84 This confirms that use of the E1371Q background effectively isolates Cl- :Pt(NO2)4 2- interactions that occur within the channel pore from those that may involve Cl- interactions with a non-pore extracellular site. Login to comment
87 (A, B) Example macroscopic I-V relationships for E1371Q when the extracellular solution contained 150 mM SCN- (A) or 150 mM ClO4 - (B). Login to comment
99 Thus, K95Q/E1371Q is associated with weakened Pt(NO2)4 2- block (Fig. 4A-E), and I344K/E1371Q with greatly strengthened block (Fig. 4F-J). Login to comment
101 Thus Pt KD was only very weakly [Cl- ]o-sensitive in K95Q/E1371Q (Fig. 4E), but very strongly [Cl- ]o-dependent in I344K/E1371Q (Fig. 4J). Login to comment
102 The affinity, voltage dependence, and [Cl- ]o-dependence of block in E1371Q, K95Q/E1371Q and I344K/E1371Q are compared directly in Fig. 5 and Table 1. Login to comment
105 Consistent with this model, neutralization of this charge in the R334Q/E1371Q mutant abolished the relationship between intracellular Pt(NO2)4 2- block and extracellular [Cl- ] (Fig. 6A-D). Login to comment
106 Block under low [Cl- ] conditions was weakened by the R334Q mutation (Table 1; also compare results for R334Q/E1371Q in Fig. 6C with E1371Q in Fig. 2D), resulting in a significant increase in Pt KD at all voltages (P b 0.002). Login to comment
109 Since the R334Q and I344K mutations in different parts of the pore (Fig. 1) had opposite effects on the [Cl- ]o-sensitivity of Pt(NO2)4 2- block - which was practically abolished in R334Q/E1371Q (Fig. 6) but greatly increased in I344K/E1371Q (Figs. 4, 5) - these mutations were combined to generate a R334Q/I344K/E1371Q mutant. Login to comment
110 Not only did R334Q/I344K/E1371Q channels exhibit strong Pt(NO2)4 2- block (Table 1), but block was also strongly dependent on extracellular [Cl- ] (Fig. 6E-H). Login to comment
111 This confirms that the I344K mutation not only strengthens interactions with intracellular Pt(NO2)4 2- ions but also with extracellular Cl-ions. Furthermore, this effect on interactions with extracellular Cl- appears independent of the presence of a positively charged side chain at position 334: whereas the R334Q mutation abolishes Cl- -dependence of block in E1371Q channels (Fig. 6D), it does not in I344K- bearing E1371Q channels (Fig. 6H). Login to comment
112 Thus, interactions with external Cl- that are lost in R334Q/E1371Q channels are at least partially restored by the I344K mutation. Login to comment
113 To examine if the I344K mutation also resulted in altered interactions with other extracellular anions - and if this effect was also independent of R334 -Pt(NO2)4 2- block was also investigated with other extracellular anions, both in I344K/E1371Q and in R334Q/I344K/ E1371Q channels (Fig. 7). Login to comment
114 In both these channel mutants Pt(NO2)4 2- block was also weakened by other extracellular anions (Fig. 7C-H), with a similar overall apparent anion selectivity to that observed for E1371Q (Fig. 3E). Login to comment
116 First, SCN- had a very strong negative effect on Pt(NO2)4 2- block in I344K/E1371Q, increasing Pt KD(0) N600-fold relative to that with gluconate and N25-fold relative to that with Cl- (Fig. 7C, E, G). Login to comment
117 Secondly, formate was able to significantly increase Pt KD(0) relative to gluconate in both I344K/E1371Q (Fig. 7G) and in R334Q/I344K/E1371Q (Fig. 7H), unlike in E1371Q where formate had no significant effect (Fig. 3D, E). Login to comment
119 A quantitative model of anion binding In constitutively open E1371Q channels, extracellular Cl- (Fig. 2) and other monovalent anions (Fig. 3) weaken the blocking effects of Pt(NO2)4 2- , suggesting that these anions compete for binding inside the channel pore. Login to comment
123 Fits to mean data for E1371Q using Eq. (3) (Fig. 8A) suggest, at 0 mV membrane potential, a Pt Kvac of 245 bc;M (Fig. 8D), Pt Kocc of 1440 bc;M (Fig. 8E), and Cl K 179 mM (Fig. 8F), with voltage dependencies of these parameters suggesting Pt zb4;vac of -0.39 (Fig. 8D), Pt zb4;occ -0.63 (Fig. 8E), and Cl zb4; +0.22 (Fig. 8F). Login to comment
126 Asterisks indicate a significant difference from E1371Q (*P b 0.05; **P b 0.001). Login to comment
128 Pt KD(0) (4 mM Cl- ) (bc;M) zb4; (4 mM Cl- ) Pt KD(0) (154 mM Cl- ) (bc;M) zb4; (154 mM Cl- ) E1371Q 183.7 &#b1; 33.2 (9) -0.397 &#b1; 0.030 (9) 441.0 &#b1; 28.6 (7) -0.503 &#b1; 0.026 (7) R899Q/E1371Q 189.9 &#b1; 55.0 (6) -0.362 &#b1; 0.063 (6) 434.0 &#b1; 32.2 (7) -0.458 &#b1; 0.048 (7) K95Q/E1371Q 1110 &#b1; 172 (6)** -0.244 &#b1; 0.022 (6)* 1422 &#b1; 218 (6)** -0.193 &#b1; 0.041 (6)** I344K/E1371Q 6.92 &#b1; 1.48 (6)** -1.589 &#b1; 0.125 (6)** 164.7 &#b1; 27.5 (7)** -1.604 &#b1; 0.080 (7)** R334Q/E1371Q 1081 &#b1; 220 (4)** -0.637 &#b1; 0.106 (4)* 1112 &#b1; 144 (4)** -0.621 &#b1; 0.051 (4)* R334Q/I344K/E1371Q 39.24 &#b1; 7.94 (4)* -1.093 &#b1; 0.037 (4)** 258.3 &#b1; 30.7 (5)* -1.075 &#b1; 0.033 (5)** Fig. 4. Effect of mutations that weaken or strengthen intracellular Pt(NO2)4 2- block. Login to comment
129 (A, B, F, G) Example macroscopic I-V relationships for K95Q/E1371Q (A, B) or I344K/E1371Q (F, G) under high (154 mM; A, F) or low (4 mM; B, G) extracellular [Cl- ] conditions. In each case currents were recorded before (control) and after the addition of Pt(NO2)4 2- (at the concentrations indicated) to the intracellular solution. Login to comment
135 There was no significant difference in these parameters for K95Q/E1371Q (E) (P N 0.05). Login to comment
136 Dotted line indicates the fit to data for E1371Q (see Fig. 2H). Login to comment
140 Quantitative analysis of anion binding in mutant channels Similar analysis of Cl- -dependent Pt(NO2)4 2- block in K95Q/E1371Q and I344K/E1371Q (Fig. 8B, C) provides additional insight into the effect of mutations close to the putative Pt(NO2)4 2-binding site. Login to comment
142 Compared to E1371Q, Pt(NO2)4 2-binding was weakened in K95Q/E1371Q, both in Cl- - unoccupied (Fig. 8D) and Cl- -occupied channels (Fig. 8E), although its voltage dependence was little changed; and Cl-binding itself was only slightly weakened (Fig. 8F). Login to comment
144 In fact, since Pt(NO2)4 2- block is so weak in K95Q/E1371Q, the accuracy of estimated Cl K in this mutant is questionable. Login to comment
146 Compared to E1371Q, the intrinsic Pt(NO2)4 2-binding affinity of I344K/E1371Q is increased ~1400-fold by the addition of a second fixed positive charge in the inner vestibule, with no apparent change in the voltage dependence of binding (Fig. 8D). Login to comment
148 In fact, the impact of Cl-binding on Pt(NO2)4 2-binding affinity (the difference between Pt Kvac and Pt Kocc) appears much greater in I344K/E1371Q than in E1371Q (Fig. 9). Login to comment
149 Finally, Cl-binding affinity is higher in I334K/E1371Q (Fig. 8F; Table 2), suggesting that the I344K mutation strengthens interactions with external Cl-ions as well as with internal Pt(NO2)4 2- ions. Furthermore, the voltage-dependence of Cl-binding is greatly increased, with Cl zb4; being increased N5-fold (Fig. 8F), suggesting that external Cl-ions cross a much larger part of the transmembrane electric field to reach their binding site in this mutant. Login to comment
150 External Cl- -dependence of Pt(NO2)4 2- block is abolished in R334Q/ E1371Q (Fig. 6C, D; Fig. 10A), consistent with the positive charge at this site in the outer vestibule of the pore (Fig. 1) being crucial for interactions between the channel and extracellular Cl-ions that are required for ion:ion interactions inside the pore [33,43]. Login to comment
151 Binding of Pt(NO2)4 2- ions to R334Q/E1371Q was somewhat weaker than E1371Q (Fig. 10C; Table 2), consistent with earlier reports that mutations at R334 disrupt blocker binding in the pore inner vestibule, perhaps by a long-range conformational effect on the pore [56]. Login to comment
152 The voltage-dependence of Pt(NO2)4 2-binding is also increased in R334Q/E1371Q (Fig. 10C). Login to comment
153 However, the very low apparent Cl- affinity of R334Q/E1371Q implied by the apparent [Cl- ]-independence of Pt KD in this mutant (Figs. 6D, 10A) precluded quantitative analysis of Pt Kocc(0) and Cl K in this case; the lack of slope illustrated in the relationships in Figs. 6D and 10A imply extremely low Cl-binding affinity, consistent with ablation of the external Cl-binding site. Login to comment
154 Although the R334Q mutant abolished Cl- -dependence of Pt(NO2)4 2- block, the double pore mutant R334Q/I344K/E1371Q showed strongly Cl- -dependent block (Fig. 6E-H; Fig. 10B), suggesting that the presence of the I344K mutant restores Cl-binding that is lost in R334Q/E1371Q. Login to comment
155 Quantitative analysis of data from R334Q/I344K/ E1371Q (Fig. 10; Table 2) suggests that the presence of the second fixed positive charge in the inner vestibule at position 344 still supports strong Pt(NO2)4 2-binding (Fig. 10C), although (as for E1371Q) Pt(NO2)4 2-binding is somewhat weakened by the R334Q mutation (Fig. 10C). Login to comment
156 Binding of Pt(NO2)4 2-to Cl- -occupied channels is similar in R334Q/E1371Q and R334Q/I344K/E1371Q (Fig. 10D), suggesting that R334 plays little or no role in ion:ion interactions in mutant channels bearing a positive charge at position 344. Login to comment
157 Chloride binding also remains strong in R334Q/I344K/E1371Q, although it too is somewhat weakened by the R334Q mutation (Fig. 10E). Login to comment
158 The strong effect of external Clon Pt(NO2)4 2-binding to R334Q/I344K/E1371Q channels is illustrated in Fig. 9D. Login to comment
159 Because the effect of external SCN- on Pt(NO2)4 2- block was so dramatically altered in I344K/E1371Q (Fig. 7C, E, G), quantitative analysis of the effects of SCN- was also carried out (Fig. 11; Table 3). Login to comment
160 As for external Cl- (Fig. 8), there was a linear relationship between [SCN- ]o and Pt KD, both in E1371Q (Fig. 11A) and I344K/E1371Q (Fig. 11E), consistent with competition between SCN- and Pt(NO2)4 2- ions. Login to comment
162 (A) Relationship between Pt(NO2)4 2-binding affinity (Pt KD at 0 mV) and [Cl- ]o for E1371Q (black), K95Q/E1371Q (blue; see Fig. 1) and I344K/E1371Q (red; see Fig. 1). Login to comment
163 Asterisks indicate a significant difference from E1371Q (P b 0.001). Login to comment
165 Asterisks and daggers indicate a significant difference from E1371Q (asterisks, P b 0.005; daggers, P b 0.0000005). Login to comment
168 (A, B, E, F) Example macroscopic I-V relationships for R334Q/E1371Q (A, B) or R334Q/I344K/E1371Q (E, F) under high (154 mM; A, E) or low (4 mM; B, F) extracellular [Cl- ] conditions. In each case currents were recorded before (control) and after the addition of Pt(NO2)4 2- (at the concentrations indicated) to the intracellular solution. Login to comment
172 There was no significant difference in these parameters for R334Q/E1371Q (E) (P N 0.5). Login to comment
173 Dotted lines indicate the fit to data for E1371Q (see Fig. 2H) or I344K/E1371Q (see Fig. 4J) as indicated. Login to comment
175 E1371Q, SCN K was greatly reduced (Fig. 11F; Table 3), Pt Kocc(0) was slightly increased (Fig. 11G; Table 3), and both SCN zb4; and Pt zb4;occ were increased (Fig. 11F, G; Table 3). Login to comment
176 In both E1371Q (Fig. 11D) and I344K/ E1371Q (Fig. 11H), SCN- binding had a greater destabilizing impact on Pt(NO2)4 2-binding than did Cl- . Login to comment
183 (A, B) Example macroscopic I-V relationships for I344K/E1371Q (A) or R334Q/I344K/E1371Q (B) with extracellular solution containing 150 mM SCN- . Login to comment
197 The present study using constitutively active E1371Q channels appears to effectively isolate the effects of Cl- acting inside the pore, at least for the test blocker Pt(NO2)4 2- . Login to comment
198 Thus, block by cytoplasmic Pt(NO2)4 2- ions - and its dependence on extracellular [Cl- ] - is independent of the R899Q mutation (in an E1371Q background) (Fig. 2; Table 1). Login to comment
208 The ability of the E1371Q background effectively to isolate antagonistic interactions between external anions and intracellular Pt(NO2)4 2- blocking anions that occur inside the open channel pore affords a novel opportunity to investigate the nature and molecular bases of these antagonistic interactions. Login to comment
216 (A-C) Effect of extracellular [Cl- ] ([Cl- ]o) on the measured Pt KD in E1371Q (A), K95Q/E1371Q (B) and I344K/E1371Q (C) at different membrane potentials as indicated in panel A. Straight-line fits to the data are to Eq. (3) as described in the Materials and methods. Login to comment
219 Data for I344K/E1371Q at positive voltages appear unreliable, likely due to the very strong voltage dependence of block observed in this mutant. Login to comment
221 Because of the lack of [Cl- ]o-dependence in R334Q/E1371Q, values for Pt Kocc, Pt zb4;occ, Cl K and Cl zb4; could not be obtained for this mutant. Login to comment
222 Pt Kvac(0) (bc;M) Pt zb4;vac Pt Kocc(0) (bc;M) Pt zb4;occ Cl K (mM) Cl zb4; E1371Q 245 -0.39 1440 -0.63 179 +0.22 K95Q/E1371Q 1150 -0.23 4400 -0.58 296 +0.20 I344K/E1371Q 0.174 -0.42 978 -1.12 0.264 +1.09 R334Q/E1371Q 1120 -0.71 - - - - R334Q/I344K/E1371Q 31.8 -1.04 1500 -1.15 232 +0.23 Fig. 9. Effect of bound extracellular Cl-ions on the binding of intracellular Pt(NO2)4 2- ions. Login to comment
224 As noted in the legend to Fig. 8, data for I344K/E1371Q at positive voltages appear unreliable, likely due to the very strong voltage dependence of block observed in this mutant. Login to comment
239 A second mechanism of knock-off in I344K-containing channel pores Addition of a second positive charge lining the inner vestibule in I344K/E1371Q increases intrinsic Pt(NO2)4 2-binding affinity ~1400-fold (Figs. 5, 8; Table 2), likely because the additional positive charge increases electrostatic interactions with divalent Pt(NO2)4 2- anions [47] (Fig. 13B). Login to comment
242 (A, B) Effect of extracellular [Cl- ] ([Cl- ]o) on the measured Pt KD in R334Q/E1371Q (A) and R334Q/I344K/E1371Q (B) at different membrane potentials as indicated. Login to comment
246 Note that, because of the apparent lack of effect of external Clon Pt(NO2)4 2- block in R334Q/ E1371Q, it was not possible to obtain values for Pt Kocc or Cl K for this mutant. Login to comment
248 As noted in the legend to Fig. 8, data for I344K/E1371Q at positive voltages appear unreliable, likely due to the very strong voltage dependence of block observed in this mutant. Login to comment
251 (A, E) Effect of extracellular [SCN- ] ([SCN- ]o) on the measured Pt KD in E1371Q (A) and I344K/E1371Q (E) at 0 mV and -100 mV membrane potential. Login to comment
255 Dotted lines in F and G indicate the fit to data for E1371Q (from B and C). Login to comment
256 As noted in the legend to Fig. 8, data for I344K/E1371Q at positive voltages appear unreliable, likely due to the very strong voltage dependence of block observed in this mutant. Login to comment
266 However, the R334Q mutation has only a minor impact on Cl-binding in the presence of I344K, and Cl-binding to the R334Q/I344K/E1371Q mutant remains strong and strongly voltage-dependent (Fig. 10; Table 2). Login to comment
267 This suggests that in I344K-containing channels (unlike E1371Q background channels), significant binding of extracellular Cl- occurs at a site that is independent of R334. Login to comment
268 Indeed, knock-off of Pt(NO2)4 2- by Cl- appears similar in I344K/ E1371Q and R334Q/I344K/E1371Q channels (Fig. 10D), suggesting that anion binding near R334 plays little role in ion:ion interactions that occur in channels bearing the I344K mutation. Login to comment
272 Not only the binding affinity of external Cl- and internal Pt(NO2)4 2- , but also the Table 3 Mean parameters obtained from analysis of the [SCN- ]o-dependence of Pt(NO2)4 2- block in E1371Q and I344K/E1371Q, as described in Fig. 11. Login to comment
274 Pt Kocc(0) (bc;M) Pt zb4;occ SCN K (bc;M) SCN zb4; E1371Q 17,500 -0.54 14,000 +0.137 I344K/E1371Q 32,900 -0.94 1.95 +0.917 Fig. 12. Login to comment
282 strengthoftheinteractionbetweenthem,isalteredinI344K-containingchan- nels.Thus,thedestabilizingeffectofCl- onPt(NO2)4 2-binding-evaluated fromthedifferencebetweenPt(NO2)4 2- bindinginvacantandCl- -occupied channels(i.e.betweenPt KvacandPt Kocc)-ismuchgreaterinI344K/E1371Q (and to a lesser extent R334Q/I344K/E1371Q) than in E1371Q or K95Q/ E1371Q(Fig.9).Strengthenedinteractionsbetweenboundanionsarealso suggestedbytheincreasedapparentcouplingbetweenthemovementof Pt(NO2)4 2- andCl- ionsinsidetheporeinI344K(Fig.8E). Login to comment
283 One model that appears consistent with these results is that addition of a second positive charge in the inner vestibule (in I344K/E1371Q) increases the number of anions that can bind within this region of the pore (Fig. 13B). Login to comment
295 Furthermore, because this destabilizing effect involves local interactions between bound anions (Fig. 13B), it may be relatively independent of anion binding in the outer vestibule of the pore - close to R334, which is required for normal anion:anion interactions observed in E1371Q background channels (Fig. 13A). Login to comment
300 Understanding the observed properties of Pt(NO2)4 2- block of I344K channels Block of I344K/E1371Q by cytoplasmic Pt(NO2)4 2-was previously described as being of very high affinity, very strong voltage dependence, and very high sensitivity to extracellular [Cl- ] [47]. Login to comment