ABCMdb

ABCC7 p.Thr351Cys

ClinVar:	c.1052C>G , p.Thr351Ser ? , not provided
CF databases:	c.1052C>T , p.Thr351Ile (CFTR1) ? , This mutation was identified in Polish infant during CF screening program. No other mutation was found after sequencing exons: 7,10,11,13,21. Mutations 3849+10kbC>T, dele2,3(21kb) and R117H were also excluded.
Predicted by SNAP2:	A: D (91%), C: D (91%), D: D (91%), E: D (95%), F: D (95%), G: D (91%), H: D (91%), I: D (95%), K: D (95%), L: D (95%), M: D (85%), N: D (91%), P: D (95%), Q: D (95%), R: D (95%), S: D (66%), V: D (91%), 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, R: N, S: N, V: N, W: N, Y: N,

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[hide] The DeltaF508 mutation disrupts packing of the tra... J Biol Chem. 2004 Sep 17;279(38):39620-7. Epub 2004 Jul 21. Chen EY, Bartlett MC, Loo TW, Clarke DM
The DeltaF508 mutation disrupts packing of the transmembrane segments of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2004 Sep 17;279(38):39620-7. Epub 2004 Jul 21., 2004-09-17 [PMID:15272010]

Abstract [show]
The most common mutation in cystic fibrosis (deletion of Phe-508 in the first nucleotide binding domain (DeltaF508)) in the cystic fibrosis transmembrane conductance regulator (CFTR) causes retention of the mutant protein in the endoplasmic reticulum. We previously showed that the DeltaF508 mutation causes the CFTR protein to be retained in the endoplasmic reticulum in an inactive and structurally altered state. Proper packing of the transmembrane (TM) segments is critical for function because the TM segments form the chloride channel. Here we tested whether the DeltaF508 mutation altered packing of the TM segments by disulfide cross-linking analysis between TM6 and TM12 in wild-type and DeltaF508 CFTRs. These TM segments were selected because TM6 appears to line the chloride channel, and cross-linking between these TM segments has been observed in the CFTR sister protein, the multidrug resistance P-glycoprotein. We first mapped potential contact points in wild-type CFTR by cysteine mutagenesis and thiol cross-linking analysis. Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wild-type background. The disulfide cross-linking occurs intramolecularly and was reducible by dithiothreitol. Introduction of the DeltaF508 mutation into these cysteine mutants, however, abolished cross-linking. The results suggest that the DeltaF508 mutation alters interactions between the TM domains. Therefore, a potential target to correct folding defects in the DeltaF508 mutant of CFTR is to identify compounds that promote correct folding of the TM domains.

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56 TM6 point mutations (M348C, T351C, and W356C) were generated in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; TM12 point mutations (T1142C and W1145C) were generated in the EcoRV (bp 2996) 3 EcoRI (bp 3643) fragment; the ⌬F508 mutation was generated in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment. Login to comment
146 Three positive cross-linking mutants, M348C/T1142C, T351C/T1142C, and W356C/W1145C were identified (see Fig. 3B, band X) and selected for further study. Login to comment
148 Fig. 2B shows the expression of WT CFTR, the single cysteine mutants M348C, T351C, W356C, T1142C, and W1145C, and the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C. Login to comment
150 The cross-linking patterns of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C showed differences when treated with different cross-linkers. Login to comment
152 Mutant T351C/T1142C, on the other hand, shows extensive cross-linking with M8M but not with M5M or M17M. Login to comment
153 It is interesting to note that both M348C and T351C in TM6 showed cross-linking to T1142C in TM12. Login to comment
158 An example of a mutant that did not show cross-linking, T351C/L1143C, is shown in Fig. 3B. Login to comment
159 Because the cross-linkable mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C also contained the 18 endogenous cysteines, it was important to test whether any of the single M348C, T351C, W356C, T1142C, or W1145C mutants showed evidence of cross-linking with endogenous cysteines. Login to comment
182 Despite the problems with aggregation, cross-linking analysis still appeared to be a useful assay because the putative cross-linked products were specific to the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C (Fig. 3B, band X). Login to comment
183 To ensure that band X was indeed the product of disulfide cross-linking between the introduced cysteines of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C, we added DTT after cross-linking. Login to comment
187 Each cDNA contained one of the cysteine mutations M348C, T351C, W356C, T1142C, or W1145C. Login to comment
188 It was found that co-expression of the single cysteine mutants M348C plus T1142C, T351C plus T1142C or W356C plus W1145C followed by treatment with the cross-linkers M5M, M8M, or M17M did not lead to cross-linking (formation of band X) (data not shown). Login to comment
189 This indicates that cross-linking occurs intramolecularly and not intermolecularly. To compare the inter-TMD interactions between WT and misprocessed CFTRs, the ⌬F508 mutation was introduced into the positive cross-linking double cysteine constructs M348C/ T1142C, T351C/T1142C, and W356C/W1145C. Login to comment
191 As shown in Fig. 6A, incorporation of the ⌬F508 mutation into mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C abolished cross-linking. Login to comment
196 To test whether the lack of cross-linking in the ⌬F508 series of double cysteine mutants was due to inaccessibility of thiol-reactive cross-linkers to the ER membrane, we tested whether mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C (lacking ⌬F508 mutation) would still show cross-linking then they were located in an intracellular membrane. Login to comment
197 To block trafficking of the mutants to the cell surface, we pretreated cells expressing mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C with 10 ␮g/ml brefeldin A. Login to comment
212 As shown in Fig. 6B, brefeldin A blocked processing of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C. Login to comment
215 Because the mature form of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C but not WT CFTR showed cross-linking, it was important to determine whether the mutants were still active. Login to comment
236 Both mutants T351C/T1142C and W356C/W1145C, however, exhibited ϳ40% reduction in activity compared with WT CFTR. Login to comment
248 Iodide efflux assays were performed on stable CHO cell lines expressing WT or one of the positive cross-linking double cysteine mutants (M348C/T1142C, T351C/ T1142C, and W356C/W1145C) as described under "Experimental Procedures." Login to comment
262 We were able to identify three mutants, M348C/T1142C, T351C/T1142C, and W356C/W1145C, that showed disulfide cross-linking in the mature WT background but not in the ⌬F508 background. Login to comment
263 Various control experiments were done to confirm that the mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C were indeed cross-linked through the introduced cysteines via the disulfide cross-linker. Login to comment
266 Finally, cross-linking was not observed when the cysteines in mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C were co-expressed on separate CFTR molecules. Login to comment
268 The ability to detect cross-linked products between TMD1 and TMD2 such as observed with mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C could be particularly useful in future studies to monitor dynamic changes in the molecule associated with phosphorylation or ATP binding/ hydrolysis. Login to comment

[hide] The chemical chaperone CFcor-325 repairs folding d... Biochem J. 2006 May 1;395(3):537-42. Loo TW, Bartlett MC, Wang Y, Clarke DM
The chemical chaperone CFcor-325 repairs folding defects in the transmembrane domains of CFTR-processing mutants.
Biochem J. 2006 May 1;395(3):537-42., 2006-05-01 [PMID:16417523]

Abstract [show]
Most patients with CF (cystic fibrosis) express a CFTR [CF TM (transmembrane) conductance regulator] processing mutant that is not trafficked to the cell surface because it is retained in the endoplasmic reticulum due to altered packing of the TM segments. CL4 (cytoplasmic loop 4) connecting TMs 10 and 11 is a 'hot-spot' for CFTR processing mutations. The chemical chaperone CFcor-325 (4-cyclohexyloxy-2-{1-[4-(4-methoxy-benezenesulphonyl)piperazin-1-yl]-ethy l}-quinazoline) rescued most CL4 mutants. To test if CFcor-325 promoted correct folding of the TMDs (TM domains), we selected two of the CL4 mutants (Q1071P and H1085R) for disulphide cross-linking analysis. Pairs of cysteine residues that were cross-linked in mature wild-type CFTR were introduced into mutants Q1071P and H1085R. The cross-linking patterns of the Q1071P or H1085R double cysteine mutants rescued with CFcor-325 were similar to those observed with mature wild-type double cysteine proteins. These results show that CFcor-325 rescued CFTR mutants by repairing the folding defects in the TMDs.

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22 Construction of CFTR double cysteine mutants M348C(TM6)/T1142C(TM12), T351C- (TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) was described previously [10]. Login to comment
115 Mature mutant Q1071P/ M348C(TM6)/T1142C(TM12) protein was cross-linked with Figure 6 Disulphide cross-linking analysis of CFTR processing mutants HEK-293 cells expressing mutants Q1071P/M348C(TM6)/T1142C(TM12), Q1071P/T351C- (TM6)/T1142C(TM12) and Q1071P/W356C(TM6)/W1145C(TM12) (A), mutants H1085R/ M348C(TM6)/T1142C(TM12), H1085R/T351C(TM6)/T1142C(TM12) and H1085R/W356C- (TM6)/W1145C(TM12) (B) or wild-type, mutant Q1071P or mutant H1085R (C) were incubated for 48 h with (+) or without (-) 3 µM CFcor-325. Login to comment
119 Mature mutant Q1071P/T351C- (TM6)/T1142C(TM12) protein was cross-linked with M8M and to a lesser extent with M17M, while the mature mutant Q1071P/W356C(TM6)/W1145C(TM12) protein was cross-linked with M5M, M8M and M17M (Figure 6A). 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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100 The oocytes 750 500 250 0 µS 180012006000 s IBMX MTSEA Cd 2+ DTT 200 100 0 µS 180012006000 s IBMX DTT Cd 2+ MTSEA A B C -100 -80 -60 -40 -20 0 20 40 % Change in conductance Y325C A326C L327C I328C K329C G330C I331C I332C L333C R334C K335C I336C F337C T338C T339C I340C S341C F342C WT I344C V345C R347C M348C A349C V350C T351C Q353C * * * * * Cd 2+ 1mM MTSEA 1mM D FIGURE 1. Login to comment
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] Correctors promote folding of the CFTR in the endo... Biochem J. 2008 Jul 1;413(1):29-36. Loo TW, Bartlett MC, Clarke DM
Correctors promote folding of the CFTR in the endoplasmic reticulum.
Biochem J. 2008 Jul 1;413(1):29-36., 2008-07-01 [PMID:18361776]

Abstract [show]
Cystic fibrosis (CF) is most commonly caused by deletion of a residue (DeltaF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) protein. The misfolded mutant protein is retained in the ER (endoplasmic reticulum) and is not trafficked to the cell surface (misprocessed mutant). Corrector molecules such as corr-2b or corr-4a are small molecules that increase the amount of functional CFTR at the cell surface. Correctors may function by stabilizing CFTR at the cell surface or by promoting folding in the ER. To test whether correctors promoted folding of CFTR in the ER, we constructed double-cysteine CFTR mutants that would be retained in the ER and only undergo cross-linking when the protein folds into a native structure. The mature form, but not the immature forms, of M348C(TM6)/T1142C(TM12) (where TM is transmembrane segment), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants were efficiently cross-linked. Mutations to the COPII (coatamer protein II) exit motif (Y(563)KDAD(567)) were then made in the cross-linkable cysteine mutants to prevent the mutant proteins from leaving the ER. Membranes were prepared from the mutants expressed in the absence or presence of correctors and subjected to disulfide cross-linking analysis. The presence of correctors promoted folding of the mutants as the efficiency of cross-linking increased from approx. 2-5% to 22-35%. The results suggest that correctors interact with CFTR in the ER to promote folding of the protein into a native structure.

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89 The aggregates probably formed because of cross-linking between the 18 endogenous cysteine residues as the M348C(TM6)/T1142C(TM12), T351C- (TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutations were introduced into a wild-type CFTR background. Login to comment

[hide] Atomic model of human cystic fibrosis transmembran... Cell Mol Life Sci. 2008 Aug;65(16):2594-612. Mornon JP, Lehn P, Callebaut I
Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces.
Cell Mol Life Sci. 2008 Aug;65(16):2594-612., [PMID:18597042]

Abstract [show]
We describe herein an atomic model of the outward-facing three-dimensional structure of the membrane-spanning domains (MSDs) and nucleotide-binding domains (NBDs) of human cystic fibrosis transmembrane conductance regulator (CFTR), based on the experimental structure of the bacterial transporter Sav1866. This model, which is in agreement with previous experimental data, highlights the role of some residues located in the transmembrane passages and directly involved in substrate translocation and of some residues within the intracellular loops (ICL1-ICL4) making MSD/NBD contacts. In particular, our model reveals that D173 ICL1 and N965 ICL3 likely interact with the bound nucleotide and that an intricate H-bond network (involving especially the ICL4 R1070 and the main chain of NBD1 F508) may stabilize the interface between MSD2 and the NBD1F508 region. These observations allow new insights into the ATP-binding sites asymmetry and into the molecular consequences of the F508 deletion, which is the most common cystic fibrosis mutation.

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153 Interestingly, it appears that all the CFTR mutants for which disulfide cross-linking was detected (M348C in TM6 and T1142C in TM12; T351C in TM6 and T1142C in TM12; W356C in TM6 and W1145C in TM12) line the chloride channel pore and face each other (Fig. 3A). 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
281 Note the lack of consistent results reported for F337C, S341C, I344C, R347C, T351C, R352C, and Q353C (shaded). 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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133 (A and B) Neither MTSES nor MTSET altered the current of cysless/T351C channels. Login to comment
290 For example, biochemical studies demonstrated that both M348C and T351C can be cross-linked to T1142C in TM12 (Chen et al. 2004). 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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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] 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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267 Chen et al.41 did not observe cross-linking of T351C and L1143C with any of the three cross-linking agents. 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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122 The major site of charge selectivity appears to be in the region of T351C and Q353C where the anion- to-cation selectivity rises to between 15 and 25 (Fig. 3B) (Cheung and Akabas, 1997). Login to comment
127 Arg352, which is between T351C and Q353C, appears to be a majordeterminantof the anion selectivity in this region. 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
131 Thus, ions passing from the extracellular end of the channel would first encounter Arg352, which we infer forms part of the charge-selectivity filter, before they could reach T351C or Q353C, thereby accounting for the greater anion selectivity observed at these residues. Login to comment
155 The electrical distance increases dramatically from S341C to T351C (Fig. 3A), suggesting that most of the electrical potential falls in this region of the channel. Login to comment
159 The largest electrical distances that we measured, to T351C and Q353C, was only 0.6. 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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71 Activation of T351C mutant and effect of MTSEAϩ. Login to comment
72 (A) Illustration of the activation of the CFTR-induced current in an oocyte expressing the T351C mutant under two-electrode voltage clamp. Login to comment
102 2 and 3 A for the mutant T351C. 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
123 Experiments illustrating data used to determine rates of reaction of the MTS reagents with the T351C mutant. Login to comment
129 (B) The natural log of the rate constants, k, for MTSES- (circles) and MTSETϩ (triangles) reacting with the T351C mutant are plotted as a function of voltage. Login to comment
148 In Fig. 3 B the natural log of the rate constants for the reactions of MTSES- and MTSETϩ with the mutant T351C are plotted as a function of membrane potential. Login to comment
154 The distance from the extracellular end to T351C and Q353C is significantly greater than to the other residues (P Ͻ 0.05). Login to comment
162 The major site of charge selectivity appears to be in the region of T351C and Q353C where the anion to cation selectivity rises to between 15 and 25 (Fig. 5). Login to comment
180 By measuring the relative rates of reaction of anionic and cationic MTS reagents with water-exposed cysteines in and flanking the M6 segment we have shown that a major determinant of anion selectivity occurs near the cytoplasmic end of the channel; access of the negatively charged MTSES- to T351C and Q353C is favored over the positively charged MTSETϩ (Fig. 5). Login to comment
183 Consistent with this, the reaction rate constants for the reaction of MTSES- with T351C and Q353C are larger than the rates with other channel-lining residues (Table II, column 2). Login to comment
185 The arginine that lies between T351C and Q353C, Arg352, appears to be a major determinant of the anion selectivity in this region; when cysteine is substituted for the arginine at position 352 the selectivity is similar to that observed in the rest of the channel (Fig. 5). 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
188 Thus, ions passing from the extracellular end of the channel would first encounter Arg352, which we infer forms part of the charge-selectivity filter, before they could reach T351C or Q353C; thereby accounting for the greater anion selectivity we observed at these residues. Login to comment
191 The increase in the reaction rate constants for MTSES- with the mutants T351C and Q353C (Table II, column 2) is consistent with these residues being near an anion binding site which increases the dwell time of MTSES- in this region of the channel thereby effectively increasing the reaction rate constants. Login to comment
200 The ability of the cationic MTS reagents to move past the anion-selectivity filter, i.e., to react with T351C and Q353C, is consistent with the lack of ideal anion selectivity that has been reported by others. Login to comment
210 Note the marked increase in anion selectivity at the residues T351C and Q353C. 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] 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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86 The peak current at -100 mV was -7117 ± 511 nA for the wild type, and ranged from -1709 ± 124 nA for the R347C mutant to -7709 + 700 nA for the T339C mutant (Fig. 2 A). Login to comment
87 The time for the currents to reach a steady-state level after application of the cAMP-activating solution was 20 + 1 min for wild type, and ranged from 14 + 2 min for 1344C to 51 + 4 min for T351C (Fig. 2 B). Login to comment
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
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