ABCMdb

ABCC7 p.Arg352Cys

ClinVar:	c.1054C>T , p.Arg352Trp D , Likely pathogenic c.1055G>A , p.Arg352Gln D , Pathogenic
CF databases:	c.1055G>A , p.Arg352Gln D , CF-causing ; CFTR1: This missense mutation, at nucleotide position 1187 (G to A) in exon 7, has been detected in an Italian CF patient through DGGE and direct sequencing. The mutation generates an Arg to Gln substitution (R352Q) and creates a novel DdeI restriction site in the mutated allele. This mutation has been detected in a PS patient (paternal chromosome), associated with the haplotype A; the maternal chromosome carries a still uncharacterized mutation. It was found in one of 60 non-[delta] Italian CF chromosomes. c.1054C>G , p.Arg352Gly (CFTR1) ? , c.1054C>T , p.Arg352Trp (CFTR1) ? , The mutation was detected by SSCP/heteroduplex analysis and identified by direct DNA sequencing. The mutation was seen in a boy referred by West Midlands Regional Genetics Service, and whose other CF mutation was [delta]F508. We have seen it only once in over 150 samples tested.
Predicted by SNAP2:	A: D (91%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (95%), I: D (91%), K: D (85%), L: D (91%), M: D (95%), N: D (95%), P: D (95%), Q: D (59%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, S: N, T: N, V: N, W: N, Y: N,

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[hide] Arg352 is a major determinant of charge selectivit... Biochemistry. 1999 Apr 27;38(17):5528-37. Guinamard R, Akabas MH
Arg352 is a major determinant of charge selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel.
Biochemistry. 1999 Apr 27;38(17):5528-37., 1999-04-27 [PMID:10220340]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator forms an anion-selective channel. We previously showed that charge selectivity, the ability to discriminate between anions and cations, occurs near the cytoplasmic end of the channel. The molecular determinants of charge selectivity, however, are unknown. We investigated the role of Arg352, a residue flanking the predicted cytoplasmic end of the M6 segment, in the mechanism of charge selectivity. We determined the Cl- to Na+ permeability ratio (PCl/PNa) from the reversal potential measured in a 10-fold NaCl gradient. For the wild type, PCl/PNa was 36 (range of 28-51). For the R352H mutant, PCl/PNa was dependent on cytoplasmic pH. At pH 5.4, the PCl/PNa was 33 (range of 27-41), similar to that of the wild type, but at pH 7.2, where the histidine should be largely uncharged, PCl/PNa was 3 (range of 2.9-3.1). For the R352C and R352Q mutants, PCl/PNa was 7 (range of 6-8) and 4 (range of 3.5-4.4), respectively. Furthermore, Na+ which does not carry a significant fraction of the current through the wild type is measurably conducted through R352Q. Thus, the charge of the side chain at position 352 is a strong determinant of charge selectivity. In the wild type, the positive charge on Arg352 contributes to an electrostatic potential in the channel that forms a barrier to cation permeation. Mutation of Arg352 did not alter the halide selectivity sequence. Selectivity among halides must involve other residues.

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101 In cell-attached patches, with 140 mM NaCl in the pipet, the single-channel conductances (in picosiemens) were 6.0 ( 0.3 for the wild type (n ) 7), 5.3 ( 0.3 for R352C (n ) 11), 4.2 ( 0.1 for R352Q (n ) 10), 4.0 ( 0.2 for R352H (n ) 4), and 5.7 ( 0.2 for Q353C (n ) 8). Login to comment
106 In cells expressing the R352C mutation, active channels were observed in about half of the cell-attached patches; 0.6 ( 0.3 (n ) 12 patches) channel was observed before activation, and 4.9 ( 0.8 channels were observed after application of the cAMP-activating reagents. Login to comment
110 Following activation with cAMP, cells transfected with wild-type CFTR had a conductance of 12 ( 2 nS (n ) 6) and cells transfected with R352C had a conductance of 2 ( 0.3 nS (n ) 8) (Figure 2A). Login to comment
113 The single-channel conductances (in picosiemens) were 6.2 ( 0.5 for the wild type (n ) 6)2 (Figure 3B), 5.9 ( 0.3 for R352C (n ) 5), 4.2 ( 0.1 for R352Q (n ) 8), and 5.7 ( 0.3 for Q353C (n ) 8). Login to comment
129 (A) Average whole-cell current-voltage relationships obtained from cells expressing either wild-type CFTR (b) or the R352C mutant (O) in symmetrical Cl--containing solutions. Login to comment
130 Note the much lower level of current in the R352C mutant than in the wild type. Login to comment
131 (B) Average single-channel current-voltage relationships for the wild type (O) and the R352C (0), R352Q (3), and R352H (bath pH of 7.2) (]) mutants. Login to comment
132 The symbols for the wild type and R352C are indistinguishable at several voltages. Login to comment
164 These uncharged substitutions also shifted the reversal potential by an amount comparable to that observed by deprotonating R352H; Erev ) -34.9 ( 2 mV (n ) 7) for R352C, and Erev ) -26.3 ( 1.9 mV (n ) 9) for R352Q (Figure 6), resulting in calculated PCl/PNa ratios of 7 (range of 6-8) and 4 (range of 3.5-4.4), respectively. Login to comment
200 Similar reversal potentials were observed for the R352C mutant: 7 ( 1 mV (n ) 8) for Br- , -13 ( 1 mV (n ) 6) for I- , and -27 ( 2 mV (n ) 5) for F- . Login to comment
209 (A and C) Average single-channel current-voltage relationships for the R352C (A) and R352Q (C) mutants in symmetrical 140 mM NaCl (2) and in the presence of the 10-fold NaCl gradient (O) as described in the legend of Figure 3A. Login to comment
212 (B and D) Single-channel recordings are shown for the R352C (B) and R352Q (D) mutants. Login to comment
217 For the R352C mutant, the conductances were similar to that of the wild type (5.9 ( 0.3 pS for Cl-, 5.2 ( 0.3 pS for Br-, 5.7 ( 0.2 pS for F-, and 3.6 ( 0.2 pS for I-). Login to comment
218 The mechanism for the decreased I- conductance compared to those of the other halide ions for both WT and R352C is not known, although in the R347D mutant the effect is eliminated (2). Login to comment
236 Table 1: Halide Permeability Ratios (PX/PCl)a bromide chloride iodide fluoride wild type 1.39 ( 0.05 1 0.71 ( 0.02 0.25 ( 0.04 R352C 1.04 ( 0.04 1 0.54 ( 0.05 0.22 ( 0.03 a Permeability ratios for the halides relative to those for Cl- for the wild type (WT) and the R352C mutant. Login to comment
248 We do know, however, that Arg352 is in the channel lining because a cysteine substituted for Arg352 reacted with charged, sulfhydryl-specific MTS reagents (19) and the reaction rates were voltage-dependent (21). Login to comment
261 This is unlikely to be the mechanism in CFTR because Arg352 does not appear to form a high-affinity Cl-binding site because the mutation R352C does not alter the halide selectivity sequence. Login to comment

[hide] CFTR: covalent and noncovalent modification sugges... J Gen Physiol. 2001 Oct;118(4):407-31. Smith SS, Liu X, Zhang ZR, Sun F, Kriewall TE, McCarty NA, Dawson DC
CFTR: covalent and noncovalent modification suggests a role for fixed charges in anion conduction.
J Gen Physiol. 2001 Oct;118(4):407-31., [PMID:11585852]

Abstract [show]
The goal of the experiments described here was to explore the possible role of fixed charges in determining the conduction properties of CFTR. We focused on transmembrane segment 6 (TM6) which contains four basic residues (R334, K335, R347, and R352) that would be predicted, on the basis of their positions in the primary structure, to span TM6 from near the extracellular (R334, K335) to near the intracellular (R347, R352) end. Cysteines substituted at positions 334 and 335 were readily accessible to thiol reagents, whereas those at positions 347 and 352 were either not accessible or lacked significant functional consequences when modified. The charge at positions 334 and 335 was an important determinant of CFTR channel function. Charge changes at position 334--brought about by covalent modification of engineered cysteine residues, pH titration of cysteine and histidine residues, and amino acid substitution--produced similar effects on macroscopic conductance and the shape of the I-V plot. The effect of charge changes at position 334 on conduction properties could be described by electrodiffusion or rate-theory models in which the charge on this residue lies in an external vestibule of the pore where it functions to increase the concentration of Cl adjacent to the rate-limiting portion of the conduction path. Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability. The results are consistent with the hypothesis that in wild-type CFTR, R334 occupies a position where its charge can influence the distribution of anions near the mouth of the pore.

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25 R347C and R352C were gifts of M. Akabas (Albert Einstein College of Medicine, Bronx, NY) and used in their parent vector, pMN. Login to comment
107 The Function of R334C and K335C CFTR Was Modified by External MTSES or MTSET but the Function of R347C and R352C CFTR Was Not Modified by these Polar Thiol Reagents Fig. 3 summarizes the results of experiments in which MTSES, MTSET, or MTSEA (100 ␮M-10 mM) were added to the solution bathing oocytes expressing wt, R334C, K335C, R347C, or R352C CFTR. Login to comment
129 In the case of R352C CFTR, our observations also differed substantially from those reported by Cheung and Akabas (1996, 1997). Login to comment
131 In contrast, we were not able to detect effects of this magnitude applying either MTSES or MTSET to oocytes expressing R352C CFTR (Fig. 5 A). Login to comment
133 Comparison of the effects of MTSES, MTSET, and MTSEA on the conductance of oocytes expressing R334C, K335C, R347C, or R352C CFTR. Login to comment
140 MTSET and MTSES, polar thiol reagents, did not produce a discernible alteration in conductance of oocytes expressing R352C CFTR. Login to comment
144 (B) Addition of 1 mM MTSEA produced a sixfold increase in conductance of an oocyte expressing R352C CFTR not previously exposed to MTSET. Login to comment
151 R352C CFTR Function Is Modified by Addition of 1 mM MTSEA to the Perfusate, However the Effect Is Not Cysteine-specific Shown in Fig. 5 B is the result of a representative exper- iment in which an oocyte expressing R352C CFTR was exposed to 1 mM MTSEA; the result being a sixfold increase in conductance. Login to comment
186 In view of the results obtained with R352C CFTR, we tested for effects of exposure to MTSET and MTSES using constructs bearing substitutions other than cysteine for R334. Login to comment
435 Even if we allow for the possibility of some modest accessibility of R352C, it is evident that the outer sites are highly reactive, whereas the inner sites are either not at all reactive or much less so. Login to comment
442 This is an important point because in the course of our experiments (e.g., R352C CFTR) and those of others using cysteine accessibility or pH titration (Coulter et al., 1995; Akabas, 1998), it has become apparent that structural changes can render sites "distant" from the mutation accessible or titratable. Login to comment

[hide] Positive charges at the intracellular mouth of the... J Gen Physiol. 2006 Nov;128(5):535-45. Epub 2006 Oct 16. Aubin CN, Linsdell P
Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel.
J Gen Physiol. 2006 Nov;128(5):535-45. Epub 2006 Oct 16., [PMID:17043152]

Abstract [show]
Many different ion channel pores are thought to have charged amino acid residues clustered around their entrances. The so-called surface charges contributed by these residues can play important roles in attracting oppositely charged ions from the bulk solution on one side of the membrane, increasing effective local counterion concentration and favoring rapid ion movement through the channel. Here we use site-directed mutagenesis to identify arginine residues contributing important surface charges in the intracellular mouth of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. While wild-type CFTR was associated with a linear current-voltage relationship with symmetrical solutions, strong outward rectification was observed after mutagenesis of two arginine residues (R303 and R352) located near the intracellular ends of the fifth and sixth transmembrane regions. Current rectification was dependent on the charge present at these positions, consistent with an electrostatic effect. Furthermore, mutagenesis-induced rectification was more pronounced at lower Cl(-) concentrations, suggesting that these mutants had a reduced ability to concentrate Cl(-) ions near the inner pore mouth. R303 and R352 mutants exhibited reduced single channel conductance, especially at negative membrane potentials, that was dependent on the charge of the amino acid residue present at these positions. However, the very low conductance of both R303E and R352E-CFTR could be greatly increased by elevating intracellular Cl(-) concentration. Modification of an introduced cysteine residue at position 303 by charged methanethiosulfonate reagents reproduced charge-dependent effects on current rectification. Mutagenesis of arginine residues in the second and tenth transmembrane regions also altered channel permeation properties, however these effects were not consistent with changes in channel surface charges. These results suggest that positively charged arginine residues act to concentrate Cl(-) ions at the inner mouth of the CFTR pore, and that this contributes to maximization of the rate of Cl(-) ion permeation through the pore.

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112 Unfortunately, R352C Figure 6. Login to comment
181 to poor expression of the R352C mutant, and as a result it remains uncertain whether this charged amino acid contributes directly to pore surface charge. 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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218 Finally, the MTSEA reactivity was restricted to only five of twenty-six residues in and flanking TM6 in our study, whereas in the earlier study, residues F337C, S341C, I344C, R347C, T351C, R352C, and Q353C were also shown to be accessible to MTS reagents. Login to comment

[hide] Mutations at arginine 352 alter the pore architect... J Membr Biol. 2008 Mar;222(2):91-106. Epub 2008 Apr 18. Cui G, Zhang ZR, O'Brien AR, Song B, McCarty NA
Mutations at arginine 352 alter the pore architecture of CFTR.
J Membr Biol. 2008 Mar;222(2):91-106. Epub 2008 Apr 18., [PMID:18421494]

Abstract [show]
Arginine 352 (R352) in the sixth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) previously was reported to form an anion/cation selectivity filter and to provide positive charge in the intracellular vestibule. However, mutations at this site have nonspecific effects, such as inducing susceptibility of endogenous cysteines to chemical modification. We hypothesized that R352 stabilizes channel structure and that charge-destroying mutations at this site disrupt pore architecture, with multiple consequences. We tested the effects of mutations at R352 on conductance, anion selectivity and block by the sulfonylurea drug glipizide, using recordings of wild-type and mutant channels. Charge-altering mutations at R352 destabilized the open state and altered both selectivity and block. In contrast, R352K-CFTR was similar to wild-type. Full conductance state amplitude was similar to that of wild-type CFTR in all mutants except R352E, suggesting that R352 does not itself form an anion coordination site. In an attempt to identify an acidic residue that may interact with R352, we found that permeation properties were similarly affected by charge-reversing mutations at D993. Wild-type-like properties were rescued in R352E/D993R-CFTR, suggesting that R352 and D993 in the wild-type channel may interact to stabilize pore architecture. Finally, R352A-CFTR was sensitive to modification by externally applied MTSEA+, while wild-type and R352E/D993R-CFTR were not. These data suggest that R352 plays an important structural role in CFTR, perhaps reflecting its involvement in forming a salt bridge with residue D993.

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98 Previous studies showed that both R347C and R352C either were not accessible to membrane-impermeant MTS reagents (methanethiosulfonate ethyltrimethylammonium [MTSET+ ] or methanethiosulfonate ethylsulfonate [MTSES- ]) applied to the extracellular solution or lacked significant functional consequences when modified; this suggested that both sites either were at the predicted cytoplasmic end of the pore, and therefore cytoplasmic to the narrow region, or were not pore-facing residues (Smith et al. 2001). Login to comment
99 R352C was, however, sensitive to membrane-permeant MTSEA+ . Login to comment
229 We previously reported that a cysteine engineered at R352 either was not accessible to the membrane-impermeable reagents MTSES- and MTSET+ applied externally or lacked significant functional consequences when modified, although R352C-CFTR (but not WT-CFTR) did respond to prolonged exposure to the membrane-permeant MTSEA+ (Smith et al. 2001). Login to comment
230 Surprisingly, similar results were found in R352Q-CFTR, suggesting that the response to MTSEA+ in R352C-CFTR was nonspecific, not being due to modification of that engineered cysteine. Login to comment
263 Designation of R352 as the anion selectivity filter was predominantly based upon differences in the rates of modification of engineered cysteines at this site (in R352C-CFTR) by positively and negatively charged sulfhydryl modifying reagents, MTSET+ and MTSES- (Cheung and Akabas 1997; Guinamard and Akabas 1999). Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 2009 Oct 27;48(42):10078-88. Alexander C, Ivetac A, Liu X, Norimatsu Y, Serrano JR, Landstrom A, Sansom M, Dawson DC
Cystic fibrosis transmembrane conductance regulator: using differential reactivity toward channel-permeant and channel-impermeant thiol-reactive probes to test a molecular model for the pore.
Biochemistry. 2009 Oct 27;48(42):10078-88., 2009-10-27 [PMID:19754156]

Abstract [show]
The sixth transmembrane segment (TM6) of the CFTR chloride channel has been intensively investigated. The effects of amino acid substitutions and chemical modification of engineered cysteines (cysteine scanning) on channel properties strongly suggest that TM6 is a key component of the anion-conducting pore, but previous cysteine-scanning studies of TM6 have produced conflicting results. Our aim was to resolve these conflicts by combining a screening strategy based on multiple, thiol-directed probes with molecular modeling of the pore. CFTR constructs were screened for reactivity toward both channel-permeant and channel-impermeant thiol-directed reagents, and patterns of reactivity in TM6 were mapped onto two new, molecular models of the CFTR pore: one based on homology modeling using Sav1866 as the template and a second derived from the first by molecular dynamics simulation. Comparison of the pattern of cysteine reactivity with model predictions suggests that nonreactive sites are those where the TM6 side chains are occluded by other TMs. Reactive sites, in contrast, are generally situated such that the respective amino acid side chains either project into the predicted pore or lie within a predicted extracellular loop. Sites where engineered cysteines react with both channel-permeant and channel-impermeant probes occupy the outermost extent of TM6 or the predicted TM5-6 loop. Sites where cysteine reactivity is limited to channel-permeant probes occupy more cytoplasmic locations. The results provide an initial validation of two, new molecular models for CFTR and suggest that molecular dynamics simulation will be a useful tool for unraveling the structural basis of anion conduction by CFTR.

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52 We proposed that these spontaneous changes, that are not seen in either wt or Cys-less CFTR, reflect the coordination of trace Table 1: Percent Change in Oocyte Conductance in the Presence of Compounda MTSETþ MTSES- [Ag(CN)2]- [Au(CN)2]- G330C O O O O I331C -51.6 ( 6.3 -28.9 ( 2.1 -63.1 ( 8.8 O I332C O O O O L333C -58.5 ( 4.8 -47.5 ( 7.6 -83.1 ( 2.2 O R334C þ76.9 ( 11.3 -84.4 ( 1.5 -67.4 ( 7.4 -41.4 ( 3.1 K335C þ10.7 ( 2.4 -37.3 ( 1.5 -29.1 ( 6.4 -54.6 ( 4.7 I336C -54.4 ( 7.9 -75.0 ( 0.6 -81.2 ( 10.5 O F337C O O -89.6 ( 1.9 -90.1 ( 1.3 T338C -37.1 ( 3.3 -85.4 ( 2.5 -75.0 ( 5.2 -88.3 ( 1.6 T339C O O -24.5 ( 7.2 O I340C O O -93.8 ( 1.0 O S341C O O -49.3 ( 4.8 O F342C O O -84.7 ( 1.8 O C343 O O O O I344C O O -66.9 ( 9.3 -77.9 ( 2.1 V345C O O -49.1 ( 9.3 O L346C O O O O R347C O O O O M348C O O -47.9 ( 8.8 -50.1 ( 3.3 A349C O O -19.0 ( 2.0 O V350C O O O O T351C O O O O R352C O O -77.5 ( 1.3 O Q353C O O -72.6 ( 4.5 -76.7 ( 2.8 a Values are means ( SE of three or more oocytes. Login to comment
180 We had previously reported that R352C/wt CFTR was not reactive toward MTSETþ and MTSES- (7), a result consistent with the findings of Beck et al. (9). Login to comment
181 We also reported, however, that R352C/wt CFTR displayed an unusual, spontaneously reversible reaction with MTSEAþ (7). Login to comment
182 In those experiments, however, qualitatively identical, reversible reactivity toward MTSEAþ was also seen using either R352Q/wt or R352H/wt CFTR constructs, suggesting that the target of MTSEAþ was 1 of the 18 endogenous cysteines in the R352C/wt protein (7). Login to comment
184 The experiment depicted in Figure 5 demonstrates that R352C/Cys-less CFTR exhibited reactivity toward [Ag(CN)2]- . Login to comment
185 Reactivity toward [Au(CN)2]- was not seen, and experiments with R352C/ wt CFTR yielded identical results (not shown). Login to comment
186 We also confirmed that neither R352C/wt nor R352C/Cys-less exhibits reactivity toward either MTSETþ or MTSES- (not shown), contrary to the reports of Chueng and Akabas (5, 6). Login to comment
212 FIGURE 5: R352C/Cys-less CFTR was reactive toward [Ag(CN)2]- (1 mM) as judged by reversal of inhibition by adding KCN (1 mM) in the continued presence of [Ag(CN)2]- . Login to comment
241 R352C/wt CFTR is not reactive toward MTSETþ and MTSES- , but St. Aubin and Linsdell (40), Cui et al. (41), and Jordan et al. (3) identified R352 as critical for the maintenance of normal anion conduction and gating in CFTR channels. Login to comment
281 Note the lack of consistent results reported for F337C, S341C, I344C, R347C, T351C, R352C, and Q353C (shaded). Login to comment
299 The reactivity of R352C CFTR toward the channel-permeant probe, [Ag(CN)2]- , clearly implicates R352 as a "pore-lining" residue, as suggested by the effect of amino acid substitutions at this position on single-channel conductance, envisioned by St. Aubin and Linsdell (40) as contributing a positive electrostatic potential to a cytoplasmic vestibule for the pore. Login to comment

[hide] Dual roles of the sixth transmembrane segment of t... J Gen Physiol. 2010 Sep;136(3):293-309. Bai Y, Li M, Hwang TC
Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation.
J Gen Physiol. 2010 Sep;136(3):293-309., [PMID:20805575]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily that functions as a chloride channel. Previous work has suggested that the external side of the sixth transmembrane segment (TM6) plays an important role in governing chloride permeation, but the function of the internal side remains relatively obscure. Here, on a cysless background, we performed cysteine-scanning mutagenesis and modification to screen the entire TM6 with intracellularly applied thiol-specific methanethiosulfonate reagents. Single-channel amplitude was reduced in seven cysteine-substituted mutants, suggesting a role of these residues in maintaining the pore structure for normal ion permeation. The reactivity pattern of differently charged reagents suggests that the cytoplasmic part of TM6 assumes a secondary structure of an alpha helix, and that reactive sites (341, 344, 345, 348, 352, and 353) reside in two neighboring faces of the helix. Although, as expected, modification by negatively charged reagents inhibits anion permeation, interestingly, modification by positively charged reagents of cysteine thiolates on one face (344, 348, and 352) of the helix affects gating. For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. For R352C, which exhibited reduced single-channel amplitude, modifications by two positively charged reagents with different chemical properties completely restored the single-channel amplitude but had distinct effects on both the open time and the closed time. These results corroborate the idea that a helix rotation of TM6, which has been proposed to be part of the molecular motions during transport cycles in other ABC transporters, is associated with gating of the CFTR pore.

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18 For R352C, which exhibited reduced single-channel amplitude, modifications by two positively charged reagents with different chemical properties completely restored the single-channel amplitude but had distinct effects on both the open time and the closed time. Login to comment
82 7 out of the 25 mutant channels exhibited a reduced single-channel current amplitude, including, from extracellular to intracellular, R334C, K335C, F337C, T338C, S341C, R347C, and R352C (Fig. 2). Login to comment
83 The single-channel amplitude is unsolv- able in the cases of R334C, S341C, R347C, and R352C due to a limited bandwidth, whereas it is 0.2-0.3 pA for Data analysis Current traces containing fewer than three channel opening levels and lasting for >1 min were selected for single-channel kinetic analysis using a program developed by L. Csanády (2000). Login to comment
144 Fig. 7 depicts a representative recording of a patch containing a single cysless/R352C CFTR channel. Login to comment
162 The restoration of the single-channel amplitude by MTSET or MTSEA on R352C compelled us to use another strategy to assess the function of the CFTR pore before and after modification. Login to comment
163 Fig. 8 shows results obtained from patches yielding macroscopic cysless/R352C channel currents. Login to comment
167 When the membrane voltage was held at 50 mV, 50 µM glibenclamide induced 27.2 ± 1.7% (n = 10) block of the Cl current for cysless/R352C. Login to comment
171 However, when R352C was modified by MTSET, the single-channel amplitude was restored to that of the cysless/WT channel (Fig. 7 A). Login to comment
173 Interestingly, although bringing back the positive charge at this position with MTSET completely restores the single-channel amplitude, gating of MTSET-modified cysless/ R352C is not fully recovered to the level of cysless/WT channels. Login to comment
185 Because anion conduction was severely perturbed by the mutations R352C and S341C, we were not able to assess the effects of MTSES modification on the single-channel amplitude for these two constructs. Login to comment
190 For the positions 344, 345, and 348, however, single-channel recordings are not helpful because no visible current glibenclamide at the same holding potential induced stronger block in the cysless/WT channel (48.6 ± 3.0%; n = 5; Fig. 8 D), the effect of the blocker was attenuated by the R352C mutation. Login to comment
197 (A; top trace) A continuous recording showing the effects of MTSET or MTSEA on a single cysless/R352C channel. Login to comment
201 (B) Single-channel amplitude, Po, open time and closed time of MTSET- (blue) and MTSEA-modified (green) cysless/R352C channel, as determined by Gaussian fitting and kinetics analysis; n = 6. inhibition of the macroscopic mean current (Fig. 4 C) and the single-channel current in the case of the cysless/ M348C channel might be due to oxidation of the introduced cysteine to a state not reactive toward either DTT or MTS reagents. Login to comment
214 Although the exact reason is unknown, the discrepancy between the extent of Figure 8. Blocking of cysless/ R352C channels by glibenclamide before and after MTS modification. Login to comment
217 (D) The fraction of block, calculated as (1Ig/ I0) × 100% (Ig and I0 are the mean current in the presence of ATP and ATP plus glibenclamide, respectively), for cysless/WT channels (gray), cysless/R352C channels before modification (black) and after modification with MTSET (blue), and after modification with MTSEA (green). Login to comment
235 The reason for this slow reaction rate is unclear, but it could be due to a perturbation of the pore architecture by the R352C mutation (Cui et al., 2008). Login to comment
302 Second, the potency to an open-channel blocker, glibenclamide, is reduced by the charge-neutralizing mutation R352C (also see Cui et al., 2008), but was restored by charge-restoring adducts. Login to comment

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

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

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145 The voltage dependence of the reaction rates of MTS reagents with CFTR (Br0 Å Cl0 ú I0 ), Xenopus CFTR (Br0 Å I0 ú Cl0 ), and human-Xenopus CFTR chimeras in which eithercysteine mutations at these residues and single-channel studies of wild-type CFTR and the mutant R352C suggest MSD1 or MSD2 of human CFTR was replaced with the equivalent region of Xenopus CFTR (hX1-6, Br0 Å I0 úthat the selectivity of CFTR for anions over cations is determined by a site located at the intracellular end of the pore Cl0 , and hX7-12, Br0 ú Cl0 ú I0 , respectively) also suggest that sequences in MSD1 likely determine the anionthat involves the residue R352 (32, 52). Login to comment
148 Therefore, other sequences must account for the differ-end of the pore and R352C is located closer to the extracellular end of the pore than either T351C or Q353C (32). Login to comment

[hide] CFTR: mechanism of anion conduction. Physiol Rev. 1999 Jan;79(1 Suppl):S47-75. Dawson DC, Smith SS, Mansoura MK
CFTR: mechanism of anion conduction.
Physiol Rev. 1999 Jan;79(1 Suppl):S47-75., [PMID:9922376]

Abstract [show]
CFTR: Mechanism of Anion Conduction. Physiol. Rev. 79, Suppl.: S47-S75, 1999. - The purpose of this review is to collect together the results of recent investigations of anion conductance by the cystic fibrosis transmembrane conductance regulator along with some of the basic background that is a prerequisite for developing some physical picture of the conduction process. The review begins with an introduction to the concepts of permeability and conductance and the Nernst-Planck and rate theory models that are used to interpret these parameters. Some of the physical forces that impinge on anion conductance are considered in the context of permeability selectivity and anion binding to proteins. Probes of the conduction process are considered, particularly permeant anions that bind tightly within the pore and block anion flow. Finally, structure-function studies are reviewed in the context of some predictions for the origin of pore properties.

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480 Three residues, R334C, R347C, neered cysteines can react with the charged MTS reagents in the pore interior. The potential flaw in this assumptionand R352C, were also inhibited by the larger cation MTSET0 . Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 2012 Mar 20;51(11):2199-212. Epub 2012 Mar 7. Norimatsu Y, Ivetac A, Alexander C, Kirkham J, O'Donnell N, Dawson DC, Sansom MS
Cystic fibrosis transmembrane conductance regulator: a molecular model defines the architecture of the anion conduction path and locates a "bottleneck" in the pore.
Biochemistry. 2012 Mar 20;51(11):2199-212. Epub 2012 Mar 7., [PMID:22352759]

Abstract [show]
We developed molecular models for the cystic fibrosis transmembrane conductance regulator chloride channel based on the prokaryotic ABC transporter, Sav1866. Here we analyze predicted pore geometry and side-chain orientations for TM3, TM6, TM9, and TM12, with particular attention being paid to the location of the rate-limiting barrier for anion conduction. Side-chain orientations assayed by cysteine scanning were found to be from 77 to 90% in accord with model predictions. The predicted geometry of the anion conduction path was defined by a space-filling model of the pore and confirmed by visualizing the distribution of water molecules from a molecular dynamics simulation. The pore shape is that of an asymmetric hourglass, comprising a shallow outward-facing vestibule that tapers rapidly toward a narrow "bottleneck" linking the outer vestibule to a large inner cavity extending toward the cytoplasmic extent of the lipid bilayer. The junction between the outer vestibule and the bottleneck features an outward-facing rim marked by T338 in TM6 and I1131 in TM12, consistent with the observation that cysteines at both of these locations reacted with both channel-permeant and channel-impermeant, thiol-directed reagents. Conversely, cysteines substituted for S341 in TM6 or T1134 in TM12, predicted by the model to lie below the rim of the bottleneck, were found to react exclusively with channel-permeant reagents applied from the extracellular side. The predicted dimensions of the bottleneck are consistent with the demonstrated permeation of Cl(-), pseudohalide anions, water, and urea.

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345 They found that R352C CFTR exhibited unstable gating and reduced current excursions reminiscent of the subconductance states reported by Cui et al.11 Modification of R352C CFTR by either MTSET+ or MTSEA+ restored the full conductance state and appeared to stabilize the gating pattern. Login to comment

[hide] Nonintegral stoichiometry in CFTR gating revealed ... J Gen Physiol. 2012 Oct;140(4):347-59. Epub 2012 Sep 10. Jih KY, Sohma Y, Hwang TC
Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation.
J Gen Physiol. 2012 Oct;140(4):347-59. Epub 2012 Sep 10., [PMID:22966014]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) protein superfamily. Unlike most other ABC proteins that function as active transporters, CFTR is an ATP-gated chloride channel. The opening of CFTR's gate is associated with ATP-induced dimerization of its two nucleotide-binding domains (NBD1 and NBD2), whereas gate closure is facilitated by ATP hydrolysis-triggered partial separation of the NBDs. This generally held theme of CFTR gating-a strict coupling between the ATP hydrolysis cycle and the gating cycle-is put to the test by our recent finding of a short-lived, post-hydrolytic state that can bind ATP and reenter the ATP-induced original open state. We accidentally found a mutant CFTR channel that exhibits two distinct open conductance states, the smaller O1 state and the larger O2 state. In the presence of ATP, the transition between the two states follows a preferred O1-->O2 order, a telltale sign of a violation of microscopic reversibility, hence demanding an external energy input likely from ATP hydrolysis, as such preferred gating transition was abolished in a hydrolysis-deficient mutant. Interestingly, we also observed a considerable amount of opening events that contain more than one O1-->O2 transition, indicating that more than one ATP molecule may be hydrolyzed within an opening burst. We thus conclude a nonintegral stoichiometry between the gating cycle and ATP consumption. Our results lead to a six-state gating model conforming to the classical allosteric mechanism: both NBDs and transmembrane domains hold a certain degree of autonomy, whereas the conformational change in one domain will facilitate the conformational change in the other domain.

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34 When mutating the positively charged arginine at position 352 (located in the sixth transmembrane segment, TM6) to cysteine, the mutant channel (R352C-CFTR) features two distinct open states with unequal conductance (Bai et al., 2010; compare Cui et al., 2008). Login to comment
71 Fig. S1 shows the closed- time distribution for R352C-CFTR. Fig. S2 presents dwell-time distributions for O1 and O2 states. Login to comment
72 Fig. S3 demonstrates the effects of the W401F mutation on the single-channel kinetics of R352C-CFTR. Login to comment
74 R E S U L T S Unique pattern of single-channel gating transitions in Cysless/R352C-CFTR During our previous studies in scanning the pore-lining residues in the sixth transmembrane segment of TMDs (TM6), a unique feature of Cysless/R352C-CFTR caught our attentions. Login to comment
80 Consistent with this idea, ATP only induces C→O1→C transitions in E1371S/R352C-CFTR, a hydrolysis-deficient mutant. Login to comment
81 The R352C mutant channel becomes a precious tool, as it allows us to distinguish the prehydrolytic (O1) and post-hydrolytic (O2) open states and thus to "visualize" ATP hydrolysis taking place within each opening burst. Login to comment
104 Figure 2. Cysless/R352C-CFTR reveals two different open states with distinct conductance level. Login to comment
105 (A) Five representative traces and amplitude histograms from a patch that contained only one Cysless/R352C-CFTR channel. Login to comment
107 (B) Four representative traces and amplitude histograms for Cysless/R352C-CFTR recorded in a condition similar to that in A, except that both pipette and perfusion solution contain 375 mM Cl (see Materials and methods for details). Login to comment
112 R352C-CFTR channels is that a higher percentage of C→O1→O2→C transitions were present under the WT background (Table 1). Login to comment
114 To further test our hypothesis that the dominant O1→O2 transition versus O2→O1 transition is the result of ATP hydrolysis, we engineered the E1371S mutation into R352C-CFTR to abolish ATP hydrolysis (Vergani et al., 2003; Bompadre et al., 2005b) and recorded ATP-dependent opening events. Login to comment
125 They showed that in addition to O1 and O2 (named s1 and s2, respectively, in Cui et al., 2008), R352C-CFTR occasionally transits to a full conductance level that is not different from that of WT-CFTR. Login to comment
130 ATP hydrolysis drives the O1→O2 transition Although our initial observations were made with Cysless/R352C-CFTR, this unique pattern of gating transitions was also seen when we introduced the R352C mutation into the WT background (Fig. 3 A and Table 1). Login to comment
131 One notable difference between R352C- and Cysless/ Ta b l e 1 Summary of opening events by different gating patterns in three CFTR mutants Gating topology O1-O2 O1 O2 O2-O1 (O1-O2) n # Total Cysless/R352C 2.75 mM ATP 100 µM ATP 720 (45%) 663 (56%) 290 (18%) 216 (18%) 175 (11%) 137 (12%) 42 (3%) 32 (3%) 375 (23%) 128 (11%) 1,602 (100%) 1,176 (100%) R352C 2.75 mM ATP 100 µM ATP 834 (55%) 1,246 (59%) 301 (20%) 406 (19%) 173 (11%) 281 (13%) 39 (3%) 45 (2%) 169 (11%) 121 (6%) 1,516 (100%) 2,099 (100%) R352C/W401F 2.75 mM ATP 100 µM ATP 733 (44%) 1,189 (54%) 326 (19%) 367 (17%) 122 (7%) 337 (15%) 28 (2%) 60 (3%) 474 (28%) 232 (11%) 1,683 (100%) 2,185 (100%) Five different gating patterns are illustrated on the top of the table. Login to comment
140 Table 1 shows that for both Cysless/R352C and R352C mutant channels, C→O1→O2→C is the prevailing transition; thus, most gating events indeed follow the long-held one-to-one stoichiometry between the gating cycle and ATP hydrolysis cycle. Login to comment
142 A simplified scheme (Scheme 1) summarizes the idea that the gating transition pattern observed in R352C-CFTR represents a cyclic steady state in which ATP hydrolysis drives a "clockwise" movement around the state diagram (compare Richard and Miller, 1990; Gunderson and Kopito, 1995). Login to comment
144 (A) Four representative traces and amplitude histograms show the gating pattern of R352C-CFTR channel in the presence of 2.75 mM ATP. Login to comment
146 (B and C) Representative traces and amplitude histograms for R352C/E1371S-CFTR in the presence (B) or absence (C) of 2.75 mM ATP. Login to comment
149 However, these nondiscernible closures, if they exist, are not the same as the original closed state (C in Scheme 1) for the following reasons: (a) the closed state of R352C-CFTR marked with stars in Fig. 2 assumes a very long lifetime (1 s [Fig. S1] vs. 300-400 ms for WT-CFTR); thus, the probability of having this state with a lifetime of <3 ms buried in an opening burst is extremely small (<0.003); (b) for this idea to be valid, one has to propose that ATP can open this presumed brief closed channel at a rate that is two to three orders faster than it does to the original closed state; and (c) as shown in the next section, multiple rounds of O1→O2 transitions can take place within an opening burst in conditions when the O2 state is stabilized, a theme predicted by Scheme 2. Login to comment
153 This idea is first supported simply by comparing the Cysless/R352C- and R352C-CFTR results shown in Table 1. Login to comment
154 The Cysless/R352C mutant channel has an O2 state that is about four times as long as that in that hydrolysis of more than one ATP molecule does take place within an opening burst. Login to comment
163 Opening events of Cysless/R352C- (A) or R352C-CFTR (B) containing one (events 1, 3, and 4 in A, and 1-4 in B) or more (events 2 and 5 in A, and 5 in B) O1→O2 transitions. Login to comment
167 Before MESET modification, Cysless/I344C/R352Q mutant channels behaved similarly as Cysless/R352C-CFTR in the presence of ATP (Fig. 5 A). Login to comment
170 After MTSET modification of Cysless/I344C/R352Q, we indeed observed robust ATP-independent openings (Fig. 5 B) with an open lifetime of 1.03 ± 0.30 s (n = 10), the R352C-CFTR (Fig. S2). Login to comment
171 Correspondingly, the percentage of opening bursts encompassing more than one O1→O2 transition is higher in Cysless/R352C (Table 1). Login to comment
181 P < 0.05. with R352C-CFTR (Fig. 3), this double mutant also exhibits a preferred order of the gating transition. Login to comment
182 Quantitative analysis of gating events indeed demonstrates a higher percentage of gating events with reentry transitions in W401F/R352C-CFTR (Table 1). Login to comment
184 The results reveal that the R352C mutation significantly shortens the total open time (150 ms [Fig. S2] vs. 400 ms for WT-CFTR ; Vergani et al., 2003; Bompadre et al., 2005a). Login to comment
186 Prolonged O1 and O2 dwell times were also found in W401F/R352C-CFTR, but to a lesser extent. Login to comment
187 The effect of Cysless and W401F mutations in prolonging the open time of R352C mutant channels is consistent with the observations made for the same mutants in the WT background (Bai et al., 2010; Tsai et al., 2010a). Login to comment
188 D I S C U S S I O N An accidental discovery of the R352C mutation grants us the opportunity to actually "see"-in real time-ATP hydrolysis taking place during CFTR gating as the ordered transition between two distinct levels of open channel conductance (O1 and O2) indicates an input of the free energy from ATP hydrolysis. Login to comment
194 After we made our discovery with R352C-CFTR, we recorded WT-CFTR under conditions described in these early reports, but did which is approximately fivefold longer than the mean lifetime of the O2 state for Cysless/R352C-CFTR (Fig. S2). Login to comment
209 Nevertheless, the striking functional similarities between state X in Jih et al. (2012) and the O2 state in this paper prompt us to test the effect of the W401F mutation on R352C-CFTR. Fig. S3 shows a representative single-channel trace of W401F/R352C-CFTR. Login to comment
210 Compared state C is nearly 1 s for R352C-CFTR. Login to comment
228 Nonetheless, the mutant R352C does offer the advantage of observing transitions between the O1 and O2 states with a much better temporal resolution necessary for a more thorough microscopic kinetic analysis. Login to comment
255 Here, we show that mutating R352 leads to a reduced opening rate (1 s1 for R352C-CFTR vs. 2.5 s1 for WT-CFTR; Vergani et al., 2003; Bompadre et al., 2005a). Login to comment
256 Figure 7. R352C shorten the locked-open time of hydrolytic-deficient CFTR mutant. Login to comment
257 Macroscopic current traces of E1371S-CFTR (A), R352C/E1371S-CFTR (B), T1246N/E1371S-CFTR (C), and R352C/T1246N/E1371S-CFTR (D). Login to comment
259 The current relaxation was fitted with a single-exponential function resulting in the relaxation time constant for each mutant: 65.6 ± 10.1 s (n = 8) for E1371S-CFTR, 4.9 ± 0.8 s (n = 12) for R352C/ E1371S-CFTR, 7.8 ± 1.6 s (n = 7) for T1246N/E1371S-CFTR, and 2.27 ± 0.27 s (n = 6) for R352C/T1246N/E1371S-CFTR. Login to comment
261 *, P < 0.05 compared with E1371S; #, P < 0.05 between two designated data. Login to comment
266 In conclusion, capitalizing on the unique gating features of R352C-CFTR, we were able to visualize ATP hydrolysis in real time for CFTR by simply monitoring single-channel current traces. Login to comment
292 It follows that mutations that decrease the rate of NBD dimerization (CATP→CAD), e.g., T1246N in Vergani et al. (2005), or those that reduce the rate for CAD→O1 (presumably the R352C mutation), will accelerate the decay rate of macroscopic currents in hydrolysis-deficient mutants upon ATP washout. Login to comment
293 Here, in Fig. 7, we show that both R352C and T1246N mutations significantly shorten the locked-open time of the hydrolytic-deficient E1371S-CFTR (Fig. 7). Login to comment
294 Notably, this result is somewhat different from that shown in Vergani et al. (2005) in which the current relaxation is prolonged in T1246N/K1250R- CFTR. Login to comment
296 Nonetheless, the locked-open time is further shortened when combining R352C and T1246N mutations together (Fig. 7, D and E), suggesting that the two mutations affects two different kinetic steps as described above. Login to comment
33 When mutating the positively charged arginine at position 352 (located in the sixth transmembrane segment, TM6) to cysteine, the mutant channel (R352C-CFTR) features two distinct open states with unequal conductance (Bai et al., 2010; compare Cui et al., 2008). Login to comment
70 Fig. S1 shows the closed-time distribution for R352C-CFTR. Fig. S2 presents dwell-time distributions for O1 and O2 states. Login to comment
73 R E S U L T S Unique pattern of single-channel gating transitions in Cysless/R352C-CFTR During our previous studies in scanning the pore-lining residues in the sixth transmembrane segment of TMDs (TM6), a unique feature of Cysless/R352C-CFTR caught our attentions. Login to comment
79 Consistent with this idea, ATP only induces C࢐O1࢐C transitions in E1371S/R352C-CFTR, a hydrolysis-deficient mutant. Login to comment
103 Figure 2.ߓ Cysless/R352C-CFTR reveals two different open states with distinct conductance level. Login to comment
106 (B) Four representative traces and amplitude histograms for Cysless/R352C-CFTR recorded in a condition similar to that in A, except that both pipette and perfusion solution contain 375 mM Cl&#e032; (see Materials and methods for details). Login to comment
111 R352C-CFTR channels is that a higher percentage of C࢐O1࢐O2࢐C transitions were present under the WT background (Table 1). Login to comment
113 To further test our hypothesis that the dominant O1࢐O2 transition versus O2࢐O1 transition is the result of ATP hydrolysis, we engineered the E1371S mutation into R352C-CFTR to abolish ATP hydrolysis (Vergani et al., 2003; Bompadre et al., 2005b) and recorded ATP-dependent opening events. Login to comment
124 They showed that in addition to O1 and O2 (named s1 and s2, respectively, in Cui et al., 2008), R352C-CFTR occasionally transits to a full conductance level that is not different from that of WT-CFTR. Login to comment
129 ATP hydrolysis drives the O1࢐O2 transition Although our initial observations were made with Cys-less/R352C-CFTR, this unique pattern of gating transitions was also seen when we introduced the R352C mutation into the WT background (Fig. 3 A and Table 1). Login to comment
139 Table 1 shows that for both Cysless/R352C and R352C mutant channels, C࢐O1࢐O2࢐C is the prevailing transition; thus, most gating events indeed follow the long-held one-to-one stoichiometry between the gating cycle and ATP hydrolysis cycle. Login to comment
141 A simplified scheme (Scheme 1) summarizes the idea that the gating transition pattern observed in R352C-CFTR represents a cyclic steady state in which ATP hydrolysis drives a "clockwise" movement around the state diagram (compare Richard and Miller, 1990; Gunderson and Kopito, 1995). Login to comment
143 (A) Four representative traces and amplitude histograms show the gating pattern of R352C-CFTR channel in the presence of 2.75 mM ATP. Login to comment
145 (B and C) Representative traces and amplitude histograms for R352C/E1371S-CFTR in the presence (B) or absence (C) of 2.75 mM ATP. Login to comment
168 Before MESET modification, Cysless/I344C/R352Q mutant channels behaved similarly as Cysless/R352C-CFTR in the presence of ATP (Fig. 5 A). Login to comment
172 Correspondingly, the percentage of opening bursts encompassing more than one O1࢐O2 transition is higher in Cysless/R352C (Table 1). Login to comment
183 Quantitative analysis of gating events indeed demonstrates a higher percentage of gating events with reentry transitions in W401F/R352C-CFTR (Table 1). Login to comment
185 The results reveal that the R352C mutation significantly shortens the total open time (&#e07a;150 ms [Fig. S2] vs. &#e07a;400 ms for WT-CFTRÊf;; Vergani et al., 2003; Bompadre et al., 2005a). Login to comment
189 D I S C U S S I O N An accidental discovery of the R352C mutation grants us the opportunity to actually "see"-in real time-ATP hydrolysis taking place during CFTR gating as the ordered transition between two distinct levels of open channel conductance (O1 and O2) indicates an input of the free energy from ATP hydrolysis. Login to comment
195 After we made our discovery with R352C-CFTR, we recorded WT-CFTR under conditions described in these early reports, but did which is approximately fivefold longer than the mean lifetime of the O2 state for Cysless/R352C-CFTR (Fig. S2). Login to comment
211 Nevertheless, the striking functional similarities between state X in Jih et al. (2012) and the O2 state in this paper prompt us to test the effect of the W401F mutation on R352C-CFTR. Fig. S3 shows a representative single-channel trace of W401F/R352C-CFTR. Login to comment
212 Compared state C is nearly 1 s for R352C-CFTR. Login to comment
230 Nonetheless, the mutant R352C does offer the advantage of observing transitions between the O1 and O2 states with a much better temporal resolution necessary for a more thorough microscopic kinetic analysis. Login to comment
258 Figure 7.ߓ R352C shorten the locked-open time of hydrolytic-deficient CFTR mutant. Login to comment
268 In conclusion, capitalizing on the unique gating features of R352C-CFTR, we were able to visualize ATP hydrolysis in real time for CFTR by simply monitoring single-channel current traces. Login to comment
295 Here, in Fig. 7, we show that both R352C and T1246N mutations significantly shorten the locked-open time of the hydrolytic-deficient E1371S-CFTR (Fig. 7). Login to comment
298 Nonetheless, the locked-open time is further shortened when combining R352C and T1246N mutations together (Fig. 7, D and E), suggesting that the two mutations affects two different kinetic steps as described above. 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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27 By mutating residue R352 in the transmembrane domain to cysteine, they created a mutant CFTR channel (R352C-CFTR) that harbors experimentally distinguishable O1 and O2 states not seen in wild-type (WT) channel gating. Login to comment
34 A critical issue that has not been addressed by Jih et al. (2012) is to what degree the R352C mutation distorts normal molecular behaviors of CFTR. Login to comment
37 This allows a qualitative comparison of WT and R352C gating behavior. Login to comment
40 Now, the rate constants derived from recording the R352C mutant could provide constraints for the ML method, which can then be applied to examine CFTR kinetic models in an even more satisfactory detail. Login to comment
50 The authors then took the story one step further by introducing a catalysis-abolishing mutation E1371S into R352C-CFTR; these hydrolysis-deficient channels now open and close reversibly (C→O1→C and C→O2→C), indicating that ATP hydrolysis underlies a unidirectional transition from O1 to O2 (Fig. 1 B). Login to comment
58 By using the R352C mutation as a tool, now Jih et al. (2012) have the ability to directly quantify the rate constants connecting C, O1, and O2 states, and thus better characterize previously undissectible molecular events, such as ATP hydrolysis (O1→O2), at the single molecule level. Login to comment
62 (C) A single-channel event of R352C-CFTR with three conductance states, acquired with my high-resolution word editor. Login to comment
25 By mutating residue R352 in the transmembrane domain to cysteine, they created a mutant CFTR channel (R352C-CFTR) that harbors experimentally distinguishable O1 and O2 states not seen in wild-type (WT) channel gating. Login to comment
32 A critical issue that has not been addressed by Jih et al. (2012) is to what degree the R352C mutation distorts normal molecular behaviors of CFTR. Login to comment
35 This allows a qualitative comparison of WT and R352C gating behavior. Login to comment
38 Now, the rate constants derived from recording the R352C mutant could provide constraints for the ML method, which can then be applied to examine CFTR kinetic models in an even more satisfactory detail. Login to comment
48 The authors then took the story one step further by introducing a catalysis-abolishing mutation E1371S into R352C-CFTR; these hydrolysis-deficient channels now open and close reversibly (C࢐O1࢐C and C࢐O2࢐C), indicating that ATP hydrolysis underlies a unidirectional transition from O1 to O2 (Fig. 1 B). Login to comment
56 By using the R352C mutation as a tool, now Jih et al. (2012) have the ability to directly quantify the rate constants connecting C, O1, and O2 states, and thus better characterize previously undissectible molecular events, such as ATP hydrolysis (O1࢐O2), at the single molecule level. Login to comment
60 (C) A single-channel event of R352C-CFTR with three conductance states, acquired with my high-resolution word editor. 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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161 This result is most likely a reflection of an increase in the open probability of I1131C CFTR channels similar to that observed for R352C CFTR by Bai et al., (2010). Login to comment
244 This result is most likely a reflection of an increase in the open probability of I1131C CFTR channels, similar to that observed for the R352C CFTR (Bai et al., 2010). Login to comment

[hide] Probing the structural and functional domains of t... J Bioenerg Biomembr. 1997 Oct;29(5):453-63. Akabas MH, Cheung M, Guinamard R
Probing the structural and functional domains of the CFTR chloride channel.
J Bioenerg Biomembr. 1997 Oct;29(5):453-63., [PMID:9511930]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) forms an anion-selective channel involved in epithelial chloride transport. Recent studies have provided new insights into the structural determinants of the channel's functional properties, such as anion selectivity, single-channel conductance, and gating. Using the scanning-cysteine-accessibility method we identified 7 residues in the M1 membrane-spanning segment and 11 residues in and flanking the M6 segment that are exposed on the water-accessible surface of the protein; many of these residues may line the ion-conducting pathway. The pattern of the accessible residues suggests that these segments have a largely alpha-helical secondary structure with one face exposed in the channel lumen. Our results suggest that the residues at the cytoplasmic end of the M6 segment loop back into the channel, narrowing the lumen, and thereby forming both the major resistance to ion movement and the charge-selectivity filter.

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128 Thus, when cysteine is substituted for Arg352 the charge selectivity drops to the same low level that is observed in the more extracellular portion of the channel (Fig. 3B). Login to comment
129 If other residues in this region were the main determinants of anion selectivity, then, the anion selectivity of the R352C mutant should have been similar to that of the adjacent residues. Login to comment
130 Moreover, based on our measurements of electrical distance, R352C is closer to the extracellular end of the channel than is T351C or Q353C (Fig. 3A). Login to comment

[hide] Locating the anion-selectivity filter of the cysti... J Gen Physiol. 1997 Mar;109(3):289-99. Cheung M, Akabas MH
Locating the anion-selectivity filter of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel.
J Gen Physiol. 1997 Mar;109(3):289-99., [PMID:9089437]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator forms an anion-selective channel; the site and mechanism of charge selectivity is unknown. We previously reported that cysteines substituted, one at a time, for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353, in and flanking the sixth membrane-spanning segment (M6), reacted with charged, sulfhydryl-specific, methanethiosulfonate (MTS) reagents. We inferred that these residues are on the water-accessible surface of the protein and may line the ion channel. We have now measured the voltage-dependence of the reaction rates of the MTS reagents with the accessible, engineering cysteines. By comparing the reaction rates of negatively and positively charged MTS reagents with these cysteines, we measured the extent of anion selectivity from the extracellular end of the channel to eight of the accessible residues. We show that the major site determining anion vs. cation selectivity is near the cytoplasmic end of the channel; it favors anions by approximately 25-fold and may involve the residues Arg347 and Arg 352. From the voltage dependence of the reaction rates, we calculated the electrical distance to the accessible residues. For the residues from Leu333 to Ser341 the electrical distance is not significantly different than zero; it is significantly different than zero for the residues Thr351 to Gln353. The maximum electrical distance measured was 0.6 suggesting that the channel extends more cytoplasmically and may include residues flanking the cytoplasmic end of the M6 segment. Furthermore, the electrical distance calculations indicate that R352C is closer to the extracellular end of the channel than either of the adjacent residues. We speculate that the cytoplasmic end of the M6 segment may loop back into the channel narrowing the lumen and thereby forming both the major resistance to current flow and the anion-selectivity filter.

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9 Furthermore, the electrical distance calculations indicate that R352C is closer to the extracellular end of the channel than either of the adjacent residues. We speculate that the cytoplasmic end of the M6 segment may loop back into the channel narrowing the lumen and thereby forming both the major resistance to current flow and the anion-selectivity filter. Login to comment
107 We did not measure the reaction rate constants for the most extracellular residue, I331C, because we thought that it was unlikely that the reaction rates would be voltage dependent given the absence of voltage dependence at the adjacent, more cytoplasmic residues. We also did not measure the reaction rate constants for the mutants I344C and R347C because, although MTSEAϩ reacted with these residues, MTSES- and MTSETϩ did not react with these k ψ( )( )ln k Ψ 0=( )( ) zFδ RT/( )-ln ψ= t a b l e i Second-order Rate Constants for the Reaction of the MTS Reagents with the Water-exposed Cysteine Mutants k ES (M-1s-1) k EA (M-1s-1) k ET (M-1s-1) mutant -25 mV -50 mV -75 mV -25 mV -50 mV -75 mV -25 mV -50 mV -75 mV L333C 71 Ϯ 3(3) 71 Ϯ 20(2) 71 Ϯ 23(3) 320 Ϯ 89(2) 320 Ϯ 128(2) 333 Ϯ 139(3) 952 Ϯ 136(2) 1,000 Ϯ 350(2) 1,053 Ϯ 443(2) R334C 48 Ϯ 14(2) 48 Ϯ 6(3) 44 Ϯ 8(4) 145 Ϯ 32(2) 163 Ϯ 7(2) 182 Ϯ 21(3) 444 Ϯ 49(2) 454 Ϯ 124(2) 588 Ϯ 95(3) K335C 36 Ϯ 20(3) 23 Ϯ 11(3) 27 Ϯ 16(3) 222 Ϯ 80(3) 121 Ϯ 51(4) 107 Ϯ 30(3) 217 Ϯ 111(3) 235 Ϯ 28(3) 217 Ϯ 95(4) F337C 91 Ϯ 17(2) 80 Ϯ 22(3) 71 Ϯ 20(4) 222 Ϯ 74(2) 222 Ϯ 86(3) 285 Ϯ 81(3) 740 Ϯ 246(3) 740 Ϯ 82(2) 714 Ϯ 51(2) S341C 56 Ϯ 18(3) 56 Ϯ 40(2) 43 Ϯ 12(3) 93 Ϯ 6(3) 110 Ϯ 22(3) 138 Ϯ 34(3) 690 Ϯ 356(3) 556 Ϯ 246(3) 800 Ϯ 224(4) T351C 100 Ϯ 25(5) 57 Ϯ 6(3) 26 Ϯ 9(6) 146 Ϯ 30(4) 195 Ϯ 42(4) 296 Ϯ 18(3) 308 Ϯ 47(10) 392 Ϯ 78(6) 769 Ϯ 89(5) R352C 42 Ϯ 4(3) 26 Ϯ 4(5) 21 Ϯ 6(4) 105 Ϯ 76(3) 137 Ϯ 46(3) 205 Ϯ 58(2) 417 Ϯ 138(4) 800 Ϯ 128(2) 952 Ϯ 408(2) Q353C 125 Ϯ 23(4) 51 Ϯ 12(4) 42 Ϯ 8(4) 83 Ϯ 24(4) 116 Ϯ 42(4) 160 Ϯ 92(3) 189 Ϯ 48(6) 220 Ϯ 48(3) 625 Ϯ 273(4) residues and therefore we could not determine the charge selectivity at these positions.2 The reaction rate constants that we have measured are between 10-and 500-fold slower than the rates of reaction with sulfhydryls in free solution (Table II) (Stauffer and Karlin, 1994). Login to comment
134 Note that the electrical distance to the residues from L333C to S341C is close to zero and that the electrical distance to R352C is smaller than the electrical distance to the adjacent residues. Login to comment
173 We now show that based on the measured electrical distances R352C appears to be closer to the extracellular end of the channel than either of the adjacent residues. Login to comment
186 If other residues in this region were the main determinants of anion selectivity, then, the anion selectivity of the R352C mutant should have been similar to that of the adjacent residues. Login to comment
187 Based on our measurements of electrical distance, R352C is closer to the extracellular end of the channel than T351C and Q353C (Fig. 4, see below). Login to comment
192 A further suggestion that Arg352 is important in charge selectivity is the increase in the reaction rate of the cationic MTSETϩ with the R352C mutant as compared to the adjacent residues (Table II, column 3). Login to comment
193 Removing the positive charge in the R352C mutant may increase the ability of cations to enter this region near the cytoplasmic end of the channel, thereby accounting for the increase rate of reaction of MTSETϩ at R352C compared to the adjacent residues. Login to comment
234 The electrical distances from the extracellular end of the channel to these three residues, with T351C being more cytoplasmic than R352C, is also inconsistent with an ␣-helical secondary structure (Fig. 4). Login to comment

[hide] Disulphonic stilbene block of cystic fibrosis tran... J Physiol. 1996 Nov 1;496 ( Pt 3):687-93. Linsdell P, Hanrahan JW
Disulphonic stilbene block of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in a mammalian cell line and its regulation by a critical pore residue.
J Physiol. 1996 Nov 1;496 ( Pt 3):687-93., [PMID:8930836]

Abstract [show]
1. The disulphonic stilbenes 4,4'-dinitrostilbene-2,2'-disulphonic acid (DNDS) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) were shown to cause a voltage-dependent inhibition of macroscopic cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents expressed in baby hamster kidney cells when applied to the cytoplasmic face of the membrane. These compounds are known to be relatively ineffective at blocking CFTR from the extracellular side of the membrane. 2. Mutation of a positively charged arginine, previously suggested to be located in the channel pore (R347), to a negatively charged aspartate significantly reduced the affinity of block by both DNDS and DIDS, suggesting that this residue contributes to the binding site for disulphonic stilbenes. 3. It is suggested that the CFTR Cl- channel may contain a relatively large inner vestibule in which a number of large anions bind and block Cl- permeation. Arginine 347 may be involved in anion binding within this region.

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102 Since MTSET reacts covalently with cysteine residues, a very low rate of MTSET permeation would presumably be sufficient to block cysteine-substitutecl forms of CFTR such as R347C and R352C, whereas a similarly low rate of permeation byv a reversible blocker such as gluconate may not affect Cl- permeation. Login to comment

[hide] Identification of cystic fibrosis transmembrane co... Biophys J. 1996 Jun;70(6):2688-95. Cheung M, Akabas MH
Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment.
Biophys J. 1996 Jun;70(6):2688-95., [PMID:8744306]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride channel that is regulated by phosphorylation and ATP binding. Work by others suggested that some residues in the sixth transmembrane segment (M6) might be exposed in the channel and play a role in ion conduction and selectivity. To identify the residues in M6 that are exposed in the channel and the secondary structure of M6, we used the substituted cysteine accessibility method. We mutated to cysteine, one at a time, 24 consecutive residues in and flanking the M6 segment and expressed these mutants in Xenopus oocytes. We determined the accessibility of the engineered cysteines to charged, lipophobic, sulfhydryl-specific methanethiosulfonate (MTS) reagents applied extracellularly. The cysteines substituted for Ile331, Leu333, Arg334, Lys335, Phe337, Ser341, Ile344, Arg347, Thr351, Arg352, and Gln353 reacted with the MTS reagents, and we infer that they are exposed on the water-accessible surface of the protein. From the pattern of the exposed residues we infer that the secondary structure of the M6 segment includes both alpha-helical and extended regions. The diameter of the channel from the extracellular end to the level of Gln353 must be at least 6 A to allow the MTS reagents to reach these residues.

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91 Effects of MTS reagents on wild-type cysteines RESULTS in CFTR To identify the residues in and flanking the M6 membrane-spanning segment that are on the water-exposed surface of As reported previously (Akabas et al., 1994b), extracellular applications of the MTS reagents to Xenopus oocytes ex- L2j K329C L. _J *G330C 1331C 1332C L333C R334C K335C 1336C F337C T338C T339C 1340C S341C T342C C343,WT 1344C V345C L346C R347C M348C A349C V350C T351C R352C Q353C 0 2000 4000 6000 8000 0 25 50 PEAK CURRENTS (nA) TIME TO REACH PLATEAU (min) FIGURE 2 Peak CFTR-induced currents and time to reach the plateau current after stimulation with cAMP-activating reagents for 24 cysteine-substitution mutants and wild-type CFTR. Login to comment
109 Accessibility of substituted cysteines to MTSES- A 1-min application of 10 mM MTSES- significantly inhibited the CFIR-induced currents of 9 of the 24 cysteine-substituted mutants (Fig. 4 A), L333C, R334C, K335C, F337C, S341C, R347C, T351C, R352C, and Q353C. Login to comment
116 We also examined the ability of a larger, permanently positively charged reagent, MTSET+, to react with three of the mutants, R334C, R347C, and R352C, that were susceptible to MTSEA+. Login to comment
117 One-minute and 8-min applications of 1 mM MTSET+ inhibited the CFTIR-mediated current of the mutants R334C by 53 ± 6% and 52 ± 7% (n = 3); R347C by 44 ± 2% and 36 + 3% (n = 3) (Fig. 3 D); and R352C by 46 ± 11% and 54.6 ± 10.5% (n = 3). Login to comment
173 Diameter of the channel lumen The ability of MTSET+ to react with the R352C mutant indicates that it can penetrate from the extracellular end to this level. Login to comment
175 Therefore, the channel diameter from the extracellular enid to the position of R352C, near the cytoplasmic end of the M6 segment, must be at least 6 A. Login to comment
90 Effects of MTS reagents on wild-type cysteines RESULTS in CFTR To identify the residues in and flanking the M6 membrane-spanning segment that are on the water-exposed surface of As reported previously (Akabas et al., 1994b), extracellular applications of the MTS reagents to Xenopus oocytes ex- L2j K329C L. _J *G330C 1331C 1332C L333C R334C K335C 1336C F337C T338C T339C 1340C S341C T342C C343,WT 1344C V345C L346C R347C M348C A349C V350C T351C R352C Q353C 0 2000 4000 6000 8000 0 25 50 PEAK CURRENTS (nA) TIME TO REACH PLATEAU (min) FIGURE 2 Peak CFTR-induced currents and time to reach the plateau current after stimulation with cAMP-activating reagents for 24 cysteine-substitution mutants and wild-type CFTR. Login to comment
108 Accessibility of substituted cysteines to MTSES- A 1-min application of 10 mM MTSES- significantly inhibited the CFIR-induced currents of 9 of the 24 cysteine-substituted mutants (Fig. 4 A), L333C, R334C, K335C, F337C, S341C, R347C, T351C, R352C, and Q353C. Login to comment
115 We also examined the ability of a larger, permanently positively charged reagent, MTSET+, to react with three of the mutants, R334C, R347C, and R352C, that were susceptible to MTSEA+. Login to comment
171 Diameter of the channel lumen The ability of MTSET+ to react with the R352C mutant indicates that it can penetrate from the extracellular end to this level. Login to comment

[hide] Nonequilibrium gating of CFTR on an equilibrium th... Physiology (Bethesda). 2012 Dec;27(6):351-61. doi: 10.1152/physiol.00026.2012. Jih KY, Hwang TC
Nonequilibrium gating of CFTR on an equilibrium theme.
Physiology (Bethesda). 2012 Dec;27(6):351-61. doi: 10.1152/physiol.00026.2012., [PMID:23223629]

Abstract [show]
Malfunction of cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC protein superfamily that functions as an ATP-gated chloride channel, causes the lethal genetic disease, cystic fibrosis. This review focuses on the most recent findings on the gating mechanism of CFTR. Potential clinical relevance and implications to ABC transporter function are also discussed.

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155 Fortuitously, this hydrolysis-dependent O1 &#a1; O2 transition was discerned in a CFTR mutant, R352C. Login to comment
156 Single-channel recordings of R352C-CFTR revealed two distinguishable conductance states: a smaller one (O1), about one-third of the WT conductance, and a larger one (O2), about one-half of the WT conductance (8). Login to comment
161 The idea that the C &#a1; O1 &#a1; O2 &#a1; C preferred transition is driven by ATP hydrolysis is supported not only by the time asymmetry but also by the observation that the O1 &#a1; O2 transition seen in an opening burst is abolished when the hydrolysis-deficient mutation (E1371S) is engineered into the R352C background. Login to comment
162 An even more intriguing observation made with the R352C mutant channel is that, occasionally, there are opening events that consist of more than one O1 &#a1; O2 transition (boxed in FIGURE 3). Login to comment
177 Unique gating features of R352C-CFTR Five representative traces from patches that contain only one R352C-CFTR in the presence of 2.75 mM ATP. Login to comment
178 R352C-CFTR shows two distinct levels of open-state conductance: the smaller O1 and the larger O2. Login to comment
196 The data on R352C-CFTR support the notion that a partial separation of the NBD dimer does not necessarily close the gate in the TMDs, but the fact that the O1 and O2 states exhibit different single-channel amplitudes indicates that ATP hydrolysis and/or subsequent partial separation of NBDs must affect the conformation of the ion permeation pathway in TMDs. Login to comment

[hide] Vx-770 potentiates CFTR function by promoting deco... Proc Natl Acad Sci U S A. 2013 Mar 12;110(11):4404-9. doi: 10.1073/pnas.1215982110. Epub 2013 Feb 25. Jih KY, Hwang TC
Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle.
Proc Natl Acad Sci U S A. 2013 Mar 12;110(11):4404-9. doi: 10.1073/pnas.1215982110. Epub 2013 Feb 25., [PMID:23440202]

Abstract [show]
Vx-770 (Ivacaftor), a Food and Drug Administration (FDA)-approved drug for clinical application to patients with cystic fibrosis (CF), shifts the paradigm from conventional symptomatic treatments to therapeutics directly tackling the root of the disease: functional defects of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel caused by pathogenic mutations. The underlying mechanism for the action of Vx-770 remains elusive partly because this compound not only increases the activity of wild-type (WT) channels whose gating is primarily controlled by ATP binding/hydrolysis, but also improves the function of G551D-CFTR, a disease-associated mutation that abolishes CFTR's responsiveness to ATP. Here we provide a unified theory to account for this dual effect of Vx-770. We found that Vx-770 enhances spontaneous, ATP-independent activity of WT-CFTR to a similar magnitude as its effects on G551D channels, a result essentially explaining Vx-770's effect on G551D-CFTR. Furthermore, Vx-770 increases the open time of WT-CFTR in an [ATP]-dependent manner. This distinct kinetic effect is accountable with a newly proposed CFTR gating model depicting an [ATP]-dependent "reentry" mechanism that allows CFTR shuffling among different open states by undergoing multiple rounds of ATP hydrolysis. We further examined the effect of Vx-770 on R352C-CFTR, a unique mutant that allows direct observation of hydrolysis-triggered gating events. Our data corroborate that Vx-770 increases the open time of WT-CFTR by stabilizing a posthydrolytic open state and thereby fosters decoupling between the gating cycle and ATP hydrolysis cycle. The current study also suggests that this unique mechanism of drug action can be further exploited to develop strategies that enhance the function of CFTR.

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6 We further examined the effect of Vx-770 on R352C-CFTR, a unique mutant that allows direct observation of hydrolysis-triggered gating events. Login to comment
40 11 www.pnas.org/cgi/doi/10.1073/pnas.1215982110 finding of a mutant, R352C-CFTR, which allows quantification of ATP hydrolysis-triggered open-to-open transition, imparts a straightforward approach to examine how Vx-770 affects the reentry pathway. Login to comment
41 Our data with R352C-CFTR not only support the hypothesis that Vx-770 lengthens the open time of WT-CFTR by increasing the frequency of reentry, but also spawn unique targets for CFTR potentiators that could complement the action of Vx-770, a crucial step toward the ultimate goal of curing CF. Login to comment
105 Effects of Vx-770 on R352C-CFTR. Login to comment
109 Fortunately, our recent discovery of a CFTR mutant, R352C, that exhibits different single-channel conductance for the O1 and O2 states grants us a unique opportunity to directly assess the effects of Vx-770 on the lifetime of the O2 state. Login to comment
122 report (24), most of the opening bursts in R352C-CFTR contain one O1 ࢐ O2 transition; only ~10% of the openings are categorized as events with more than one O1 ࢐ O2 transition. Login to comment
141 Furthermore, by examining the effects of Vx-770 on R352C-CFTR, we were able to show that this compound indeed increases the frequency of reentry events (Fig. 4 and Table 1). Login to comment
142 Notably, the reentry pathway was first suggested by a report using PPi as a tool to fish out a unique posthydrolytic state (36) and later established by scrutinizing the R352C mutant channel that exhibits hydrolysis-dependent open channel conductance: unequal single-channel current amplitudes between pre-and posthydrolytic states (24). Login to comment
153 Effects of Vx-770 on R352C-CFTR. Login to comment
154 (A and B) Representative single-channel traces for R352C-CFTR in the absence (A) or presence (B) of 200 nM Vx-770. Login to comment
157 Summary of the effects of Vx-770 on the gating patterns exhibited in R352C-CFTR and W401F/R352C-CFTR Total Experimental condition Without Vx-770* R352C (%) 834 (55) 301 (20) 173 (11) 39 (3) 169 (11) 1,516 (100) R352C/W401F (%) 733 (44) 326 (19) 122 (7) 28 (2) 474 (28) 1,683 (100) With 200 nM Vx-770 R352C (%) 578 (57) 126 (12) 73 (7) 20 (2) 217 (21) 1,014 (100) R352C/W401F (%) 411 (37) 162 (15) 79 (7) 31 (3) 425 (38) 1,108 (100) Five different categories of the gating pattern are illustrated. Login to comment
161 *Data for R352C-CFTR and R352C/W401F-CFTR in the absence of Vx-770 were taken from ref. 24. gate in the absence of ATP, C2 ࢒ O2) primarily affected by Vx-770 are supposed to take place in CFTR`s TMDs (24). Login to comment
180 As shown in Fig. S3 and Table 1, the W401F mutation almost doubles the reentry frequency of R352C-CFTR and also significantly enhances the effect of Vx-770 (Fig. 5, Table 1 and Fig. S3). Login to comment
207 Because the single-channel conductance is reduced by the R352C mutation, a pipette solution with 375 mM Cl-was used (in millimoles): Fig. 5. Login to comment
209 Representative single-channel traces for R352C/W401F-CFTR treated with 2.75 mM ATP in the absence (A) or presence (B) of 200 nM Vx-770. Login to comment
210 Frequency of opening bursts containing multiple rounds of O1 ࢐ O2 transition (summarized in Table 1) is increased and the overall open time is prolonged (Fig. S3D) compared with R352C-CFTR recorded in the same condition. Login to comment
213 For inside-out configuration, the 150 mM Cl- perfusion solution contained (in millimoles): 150 NMDG-Cl, 2 MgCl2, 10 EGTA, and 8 Tris (pH 7.4 with NMDG), the 375 mM Cl- perfusion solution used for R352C-CFTR contained (in millimoles): 375 NMDG-Cl, 2 MgCl2, 10 EGTA, and 8 Tris (pH 7.4 with NMDG). Login to comment
223 For kinetic analysis of R352C-CFTR, three current levels (C, O1, and O2) were determined from all point histograms. Login to comment

[hide] Two salt bridges differentially contribute to the ... J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24. Cui G, Freeman CS, Knotts T, Prince CZ, Kuang C, McCarty NA
Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function.
J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24., [PMID:23709221]

Abstract [show]
Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

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72 Open burst durations and closed durations were measured from single channel recordings of WT-, R352C-, D993C-, and R352C/D993C-CFTR, and then histograms and fits of them with single exponential functions were generated with IGOR (WaveMetrics, Inc., Lake Oswego, OR) to determine time constants for open burst durations (঄o, also called beta) and closed durations (঄c, also called ॷ) for all of the constructs. Login to comment
152 We generated R352C/D993C-CFTR and exposed the channels to MTS-2-MTS; MTS-2-MTS was chosen for this experiment because it leads to cross-linking of cysteines at a distance of b03;4.6 &#c5;, which is within the average distance for known salt bridges in a varietyofproteins(13).Priortostudieswiththedoublemutant,we also investigated each single mutant and their responses to monofunctional sulfhydryl-modifying reagents. Login to comment
162 Recovery of Charge at R352C and D993C Rescued Channel Stability in the Full Open State-R352C-CFTR exhibited single channel behavior similar to that previously reported for R352A-, R352Q-, and R352E-CFTR (13). Login to comment
163 A representative recording is shown in Fig. 5A, taken from one membrane patch bearing R352C-CFTR before and after exposure to MTSEAaf9; and after wash out. Login to comment
165 Prior to exposure to MTSEAaf9; , R352C-CFTR exhibited multiple conductance states, including closed (c) and s1, s2, and f open states. Login to comment
166 R352C-CFTR channels opened to all open states for very short durations (Fig. 5B). Login to comment
167 After exposure to MTSEAaf9; , R352C-CFTR channels exhibited mainly the f state with much longer mean burst duration and appearance of the s1 and s2 states as rare events, indicating recovery of open state stability (Fig. 5B). Login to comment
171 In contrast, deposition of negative charge at R352C-CFTR by exposure to MTSESafa; did not alter channel behavior (supplemental Fig. 2A). Login to comment
174 We note that in R352C-CFTR on the WT-CFTR background, exposure to MTSEAaf9; and MTSETaf9; led to an increase in NPo (Fig. 5A), reflecting modification of endogenous cysteines. Login to comment
176 In contrast to these results for R352C-CFTR, the stability of single channel opening in D993C-CFTR was rescued to mimic that of WT-CFTR by exposure to MTSESafa; (but not MTSEAaf9; or MTSETaf9; ), leading to significantly increased mean burst duration (supplemental Fig. 3B). Login to comment
179 We repeated the above experiments in R352C/Cys-less V510A-CFTR and D993C/ Cys-less V510A-CFTR to further rule out the possibility of any endogenous cysteines being involved in the process. Login to comment
182 A Bifunctional MTS Reagent Can Latch R352C/D993C-CFTR into the Full Open State Even after Washout of ATP-We hypothesized that the CFTR channel pore could be latched into the open state by cross-linking the two cysteines at R352C and D993C. Login to comment
183 We first tested the effects of monofunctional reagents MTSETaf9; , MTSEAaf9; , and MTSESafa; on the double mutant R352C/D993C-CFTR. Login to comment
184 None of these reagents rescued salt Dynamic Modulation of the CFTR Pore by Salt Bridges JULY 12, 2013ߦVOLUME 288ߦNUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20763 bridge function to stabilize channel behavior in R352C/D993C-CFTR in terms of stable openings to the f state (data not shown). Login to comment
185 Before applying MTS-2-MTS to the R352C/D993C-CFTR double mutant, we first tested the effects of this bifunctional linker on WT-CFTR (Fig. 6A) and Cys-less V510A-CFTR (Fig. 6B). Login to comment
187 We then examined the effects of MTS-2-MTS on R352C-D993C-CFTR (on the WT-CFTR background); a representative experiment is shown in Fig. 7. Login to comment
188 In the presence of ATP and PKA, prior to the addition of MTS-2-MTS, R352C/D993C-CFTR exhibited low open probability, unstable openings to the f state, and occasional subconductance open states. Login to comment
191 It seems likely that this reflects the fact that there are several possible consequences of exposing the double mutant to MTS-2-MTS, including covalent modification of R352C and D993C separately by two MTS-2-MTS molecules within each CFTR protein. Login to comment
195 Deposition of positive charge at R352C improved stability of the open state. Login to comment
196 A, sample traces from R352C-CFTR recorded from one patch under control conditions (top trace, ATP af9; PKA) and in the presence of 100 òe;M MTSEAaf9; (middle trace, ATP af9; PKA af9; MTSEAaf9; ) and then after washout with a large volume of intracellular solution and the subsequent addition of ATP and PKA alone (bottom trace, ATP af9; PKA) in excised inside-out membrane patches. Login to comment
198 All traces were recorded at VM afd; afa;100 mV. B, comparison of mean burst duration of R352C in the absence of MTSEAaf9; (ATP af9; PKA only) (afa;MTSEA) and in the presence of MTSEAaf9; with ATP af9; PKA (af9;MTSEA). Login to comment
209 The free energy change èc;èc;G between WT-CFTR and R352C/D993C-CFTR was afa;1.508 kcal/mol, which suggests that R352C and D993C interact with each other when CFTR is in the open state (supplemental Fig. 5). Login to comment
218 A, effects of 100 òe;M MTS-2-MTS on R352C-D993C-CFTR. Login to comment
220 B, MTS-2-MTS failed to functionally modify R352C/D993C-CFTR when applied when the channel was in the closed state. Login to comment

[hide] Acute inhibition of the cystic fibrosis transmembr... Am J Physiol Cell Physiol. 2013 Oct 15;305(8):C817-28. doi: 10.1152/ajpcell.00052.2013. Epub 2013 Jun 19. Cai Z, Li H, Chen JH, Sheppard DN
Acute inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel by thyroid hormones involves multiple mechanisms.
Am J Physiol Cell Physiol. 2013 Oct 15;305(8):C817-28. doi: 10.1152/ajpcell.00052.2013. Epub 2013 Jun 19., [PMID:23784545]

Abstract [show]
The chemical structures of the thyroid hormones triiodothyronine (T3) and thyroxine (T4) resemble those of small-molecules that inhibit the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. We therefore tested the acute effects of T3, T4 and reverse T3 (rT3) on recombinant wild-type human CFTR using the patch-clamp technique. When added directly to the intracellular solution bathing excised membrane patches, T3, T4, and rT3 (all tested at 50 muM) inhibited CFTR in several ways: they strongly reduced CFTR open probability by impeding channel opening; they moderately decreased single-channel current amplitude, and they promoted transitions to subconductance states. To investigate the mechanism of CFTR inhibition, we studied T3. T3 (50 muM) had multiple effects on CFTR gating kinetics, suggestive of both allosteric inhibition and open-channel blockade. Channel inhibition by T3 was weakly voltage dependent and stronger than the allosteric inhibitor genistein, but weaker than the open-channel blocker glibenclamide. Raising the intracellular ATP concentration abrogated T3 inhibition of CFTR gating, but not the reduction in single-channel current amplitude nor the transitions to subconductance states. The decrease in single-channel current amplitude was relieved by membrane depolarization, but not the transitions to subconductance states. We conclude that T3 has complex effects on CFTR consistent with both allosteric inhibition and open-channel blockade. Our results suggest that there are multiple allosteric mechanisms of CFTR inhibition, including interference with ATP-dependent channel gating and obstruction of conformational changes that gate the CFTR pore. CFTR inhibition by thyroid hormones has implications for the development of innovative small-molecule CFTR inhibitors.

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257 Recognizing that sojourns to subconductance states in Cysless-R352C-CFTR mimic the behavior of wild-type CFTR blocked by MOPS, Jih et al. (33) developed the energetic coupling model of CFTR channel gating (for review, see Ref. 32). Login to comment

[hide] Understanding how cystic fibrosis mutations disrup... Int J Biochem Cell Biol. 2014 Jul;52:47-57. doi: 10.1016/j.biocel.2014.04.001. Epub 2014 Apr 13. Wang Y, Wrennall JA, Cai Z, Li H, Sheppard DN
Understanding how cystic fibrosis mutations disrupt CFTR function: from single molecules to animal models.
Int J Biochem Cell Biol. 2014 Jul;52:47-57. doi: 10.1016/j.biocel.2014.04.001. Epub 2014 Apr 13., [PMID:24727426]

Abstract [show]
Defective epithelial ion transport is the hallmark of the life-limiting genetic disease cystic fibrosis (CF). This abnormality is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the ATP-binding cassette transporter that functions as a ligand-gated anion channel. Since the identification of the CFTR gene, almost 2000 disease-causing mutations associated with a spectrum of clinical phenotypes have been reported, but the majority remain poorly characterised. Studies of a small number of mutations including the most common, F508del-CFTR, have identified six general mechanisms of CFTR dysfunction. Here, we review selectively progress to understand how CF mutations disrupt CFTR processing, stability and function. We explore CFTR structure and function to explain the molecular mechanisms of CFTR dysfunction and highlight new knowledge of disease pathophysiology emerging from large animal models of CF. Understanding CFTR dysfunction is crucial to the development of transformational therapies for CF patients.

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1915 However, Jih et al. (2012) interpreted the sojourns of the CFTR construct Cysless-R352C-CFTR to sub-conductance states to suggest that more than one ATP molecule might be hydrolysed during an open channel burst. Login to comment

[hide] Three charged amino acids in extracellular loop 1 ... J Gen Physiol. 2014 Aug;144(2):159-79. doi: 10.1085/jgp.201311122. Epub 2014 Jul 14. Cui G, Rahman KS, Infield DT, Kuang C, Prince CZ, McCarty NA
Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR.
J Gen Physiol. 2014 Aug;144(2):159-79. doi: 10.1085/jgp.201311122. Epub 2014 Jul 14., [PMID:25024266]

Abstract [show]
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1-6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5'-(beta,gamma-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues.

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268 To further test the possible salt bridge between R104 and E116, we made use of MTS reagents that we used previously to confirm interactions between R352C and D993C (Cui et al., 2013). Login to comment
321 This is in contrast to the ability of MTS-2-MTS to lock R352C/D993C-CFTR into the open state or to lock R334C/E217C-CFTR into the closed state (Cui et al., 2013; Rahman et al., 2013). Login to comment

[hide] Modulation of CFTR gating by permeant ions. J Gen Physiol. 2015 Jan;145(1):47-60. doi: 10.1085/jgp.201411272. Epub 2014 Dec 15. Yeh HI, Yeh JT, Hwang TC
Modulation of CFTR gating by permeant ions.
J Gen Physiol. 2015 Jan;145(1):47-60. doi: 10.1085/jgp.201411272. Epub 2014 Dec 15., [PMID:25512598]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is unique among ion channels in that after its phosphorylation by protein kinase A (PKA), its ATP-dependent gating violates microscopic reversibility caused by the intimate involvement of ATP hydrolysis in controlling channel closure. Recent studies suggest a gating model featuring an energetic coupling between opening and closing of the gate in CFTR's transmembrane domains and association and dissociation of its two nucleotide-binding domains (NBDs). We found that permeant ions such as nitrate can increase the open probability (Po) of wild-type (WT) CFTR by increasing the opening rate and decreasing the closing rate. Nearly identical effects were seen with a construct in which activity does not require phosphorylation of the regulatory domain, indicating that nitrate primarily affects ATP-dependent gating steps rather than PKA-dependent phosphorylation. Surprisingly, the effects of nitrate on CFTR gating are remarkably similar to those of VX-770 (N-(2,4-Di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide), a potent CFTR potentiator used in clinics. These include effects on single-channel kinetics of WT CFTR, deceleration of the nonhydrolytic closing rate, and potentiation of the Po of the disease-associated mutant G551D. In addition, both VX-770 and nitrate increased the activity of a CFTR construct lacking NBD2 (DeltaNBD2), indicating that these gating effects are independent of NBD dimerization. Nonetheless, whereas VX-770 is equally effective when applied from either side of the membrane, nitrate potentiates gating mainly from the cytoplasmic side, implicating a common mechanism for gating modulation mediated through two separate sites of action.

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272 Lately, by studying single-channel gating events of a CFTR mutant, R352C/Q, which exhibits unequivocal hydrolysis-dependent transitions of two distinct open states, Jih et al. (2012a) proposed an energetic coupling model featuring a more relaxed relationship between ATP hydrolysis in NBDs and opening/closing of the gate in CFTR`s TMDs. Login to comment

[hide] Cystic Fibrosis Transmembrane Conductance Regulato... J Biol Chem. 2015 Sep 18;290(38):22891-906. doi: 10.1074/jbc.M115.665125. Epub 2015 Jul 30. Corradi V, Vergani P, Tieleman DP
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR): CLOSED AND OPEN STATE CHANNEL MODELS.
J Biol Chem. 2015 Sep 18;290(38):22891-906. doi: 10.1074/jbc.M115.665125. Epub 2015 Jul 30., [PMID:26229102]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily. CFTR controls the flow of anions through the apical membrane of epithelia. Dysfunctional CFTR causes the common lethal genetic disease cystic fibrosis. Transitions between open and closed states of CFTR are regulated by ATP binding and hydrolysis on the cytosolic nucleotide binding domains, which are coupled with the transmembrane (TM) domains forming the pathway for anion permeation. Lack of structural data hampers a global understanding of CFTR and thus the development of "rational" approaches directly targeting defective CFTR. In this work, we explored possible conformational states of the CFTR gating cycle by means of homology modeling. As templates, we used structures of homologous ABC transporters, namely TM(287-288), ABC-B10, McjD, and Sav1866. In the light of published experimental results, structural analysis of the transmembrane cavity suggests that the TM(287-288)-based CFTR model could correspond to a commonly occupied closed state, whereas the McjD-based model could represent an open state. The models capture the important role played by Phe-337 as a filter/gating residue and provide structural information on the conformational transition from closed to open channel.

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268 To rationalize the effects of small, positively charged reagents when a cysteine replaces Arg-352 (72), we can hypothesize that the shorter adduct (-CH2CH2-NH3 af9; ) on the cysteine might restore a functionally important positive charge in a position similar to that present in the native arginine, whereas the bulkier adduct (-CH2CH2-N(CH3)3 af9; ) might also sterically destabilize the McjD-like open conformation, given the relative narrowing of this region of the pore. Login to comment