ABCMdb

ABCC7 p.Thr1142Cys

CF databases:	c.3425C>T , p.Thr1142Ile (CFTR1) ? , This mutation was found by DGGE and direct DNA sequencing in asthamtic patients of age older than 60 years. Reported in Hum Mut 14:510-519(1999)
Predicted by SNAP2:	A: D (63%), C: D (66%), D: D (85%), E: D (85%), F: D (80%), G: D (80%), H: D (85%), I: D (75%), K: D (91%), L: D (75%), M: D (63%), N: D (75%), P: D (85%), Q: D (80%), R: D (91%), S: N (53%), V: D (66%), W: D (91%), Y: D (85%),
Predicted by PROVEAN:	A: N, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: N, P: D, Q: D, R: D, S: N, V: N, W: D, Y: D,

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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
151 Mutant M348C/T1142C, for example, showed cross-linking with M5M and M8M but not with M17M. 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
155 Therefore, it is not surprising that the substituted cysteines at both of these positions would cross-link to the same residue, T1142C. 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
235 The M348C/T1142C mutant showed a similar level of activity as WT CFTR. 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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31 Disulphide cross-linking analysis The cDNAs of double cysteine mutants M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/ W1145C(TM12) constructed in wild-type, mutant Q1071P or mutant H1085R backgrounds were expressed in HEK-293 cells in the presence or absence of 3 µM CFcor-325. Login to comment
110 Mutants Q1071P or H1085R containing M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/ W1145C(TM12) mutations were then transiently expressed in HEK-293 cells in the presence or absence of 3 µM CFcor-325 for 48 h and then treated with the homobifunctional cross-linkers M5M, M8M or M17M. 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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103 There was a little aggregation of immature CFTR, however, when cysteine-less/V510A containing the M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/ W1145C(TM12) mutations were treated with M8M cross-linker (Figure 2B). Login to comment
124 Slow-migrating product was not detected when single-cysteine mutants M348C(TM6), T351C(TM6), W356C(TM6), T1142C(TM12) and W1145(TM12) in cysteine-less CFTR/V510A were each treated with M8M (results not shown) We then examined whether correctors, channel blockers or potentiators inhibited cross-linking of M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/ W1145C(TM12) mutants. Login to comment
178 To confirm that the corrector was modulating folding in the ER, we expressed wild-type CFTR and T351C(TM6)/T1142C- (TM12)/Y563N/cysteine-less/V510A mutant in the absence or presence of brefeldin A before cross-linking with M8M cross-linker. Login to comment
193 the double-cysteine mutants M348C(TM6)/T1142C-(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C- (TM12) in the Y563N/cysteine-less/V510A CFTR background. Login to comment
242 This delayed interaction between the TMDs might be reflected in the relatively low amount (2-4% of total CFTR) of cross-linked product in M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/ W1145C(TM12) mutants when grown in the absence of corr-4a, but is increased to 22-35% when grown in the presence of corr-4a (Figures 5D and 5E). 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] 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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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] Benzbromarone stabilizes DeltaF508 CFTR at the cel... Biochemistry. 2011 May 31;50(21):4393-5. Epub 2011 May 3. Loo TW, Bartlett MC, Clarke DM
Benzbromarone stabilizes DeltaF508 CFTR at the cell surface.
Biochemistry. 2011 May 31;50(21):4393-5. Epub 2011 May 3., 2011-05-31 [PMID:21520952]

Abstract [show]
Deletion of Phe508 from the first nucleotide-binding domain of the CFTR chloride channel causes cystic fibrosis because it inhibits protein folding. Indirect approaches such as incubation at low temperatures can partially rescue DeltaF508 CFTR, but the protein is unstable at the cell surface. Here, we show that direct binding of benzbromarone to the transmembrane domains promoted maturation and stabilized DeltaF508 CFTR because its half-life at the cell surface was ~10-fold longer than that for low-temperature rescue. Therefore, a search for small molecules that can rescue and stabilize DeltaF508 CFTR could lead to the development of an effective therapy for cystic fibrosis.

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28 It was shown that benzbromarone appeared to interact with the CFTR TMDs because 200 μM benzbromarone blocked cross-linking between cysteines introduced into TM segments 6 and 12 (M348C/T1142C).23 This concentration of benzbromarone is now shown to inhibit maturation of CFTR (Figure 1A). Login to comment
50 (C) Effect of benzbromarone on cross-linking (X-link) between cysteines in TMD1 and TMD2 (M348C/T1142C) or NBD1 and TMD2 (V510C/A1067C).7 (D) Immunoblot of cells expressing CFTR TMD1þ2 in the absence (À) or presence (þ) of 0.05 mM benzbromarone. Login to comment

[hide] Functional arrangement of the 12th transmembrane r... Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28. Qian F, El Hiani Y, Linsdell P
Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant.
Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28., [PMID:21796338]

Abstract [show]
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) alpha-helices, arranged into two pseudo-symmetrical groups of six. While TM6 in the N-terminal TMs is known to line the pore and to make an important contribution to channel properties, much less is known about its C-terminal counterpart, TM12. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of TM12 in a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM12 residues N1138, M1140, S1141, T1142, Q1144, W1145, V1147, N1148, and S1149 when applied to the cytoplasmic side of open channels. Cysteines sensitive to internal MTS reagents were not modified by extracellular [2-(trimethylammonium)ethyl] MTS, consistent with MTS reagent impermeability. Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation. Comparison of these results with previous work on CFTR-TM6 allows us to develop a model of the relative positions, functional contributions, and alignment of these two important TMs lining the CFTR pore. We also propose a mechanism by which these seemingly structurally symmetrical TMs make asymmetric contributions to the functional properties of the channel pore.

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5 Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation. Login to comment
59 a Example time courses of macroscopic currents (measured at -80 mV) carried by N1138C and T1142C as indicated in inside-out membrane patches. Login to comment
62 b Example leak subtracted I-V relationships for N1138C, T1142C, V1147C, and N1148C, recorded from inside out membrane patches following maximal channel activation with PKA, ATP, and PPi. Login to comment
64 As described in the text, whereas MTSES application always led to a decrease in macroscopic current amplitude in reactive mutants, the effects of MTSET were to decrease (e.g., N1138C, V1147C), increase (e.g., T1142C) or not significantly alter (e.g., N1148C) macroscopic current amplitude. Login to comment
80 Application of MTSES (200 μM) or MTSET (2 mM) to the intracellular solution after channel activation with PKA, ATP, and PPi significantly altered macroscopic current amplitude in nine out of 19 cysteine-substituted mutants tested (N1138C, M1140C, S1141C, T1142C, Q1144C, W1145C, V1147C, N1148C, and S1149C; Figs. 1 and 2). Login to comment
84 Closer to the intracellular end of TM12 (in T1142C, Q1144C, and W1145C), macroscopic current amplitude was decreased by MTSES application but increased by MTSET (Figs. 1 and 2). Login to comment
92 For MTS reagent-sensitive TM12 mutants located relatively deeply into the pore from its cytoplasmic end (N1138C, M1140C, S1141C, and T1142C), the rate of modification was estimated from the time course of macroscopic current amplitude change following application of MTSES (20-200 μM). Login to comment
93 As shown in Fig. 3a, modification was rapid in M1140C, S1141C, and T1142C, even using a low concentration of MTSES (20 μM), and noticeably slower in N1138C, even with 200 μM MTSES. Login to comment
106 We used a similar approach to determine whether N1138C, M1140C, S1141C, and T1142C, located relatively deeply into the pore from its cytoplasmic end and all strongly sensitive to inhibition by intracellular MTSES (Figs. 2 and 3), could be modified by MTSES pretreatment. Login to comment
118 Both S1141C and T1142C channels were again rendered insensitive to the test exposure to MTSES, again consistent with them having been covalently modified during pretreatment. Login to comment
120 These results, which are summarized quantitatively in Fig. 5d, suggest that while S1141C and T1142C can be modified by MTSES prior to channel activation, N1138C and M1140C are modified by MTSES only very slowly, if at all, in channels that have not been activated by PKA and ATP. Login to comment
140 In this respect, the slow rate of modification observed in N1138C (Fig. 3b) is similar to that we reported for P99C and L102C in TM1 [41] and T338C and S341C in TM6 [9], and the much higher modification rate constant for T1142C, S1141C, and (to a lesser extent) M1140C is closer to that reported for K95C in TM1 [41] and I344C, V345C, and M348C in TM6 [9]. Login to comment
143 a Example timecourses of macroscopic current amplitudes (measured at -50 mV) carried by N1138C, M1140C, S1141C, and T1142C as indicated in inside-out membrane patches. Login to comment
148 Using a similar approach, we find that in TM12, S1141C and T1142C can be readily modified by cytoplasmic MTSES prior to channel activation (Fig. 5), whereas N1138C and M1140C are modified rapidly after channel activation (Fig. 3) but very slowly, if at all, prior to channel activation (Fig. 5). 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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259 Chen et al.41 observed cross-linking of M348C and T1142C by M5M and M8M but not M17M. Login to comment
262 The two ends of M17M are predicted by the MD simulation to come close to each other in free solution, forming a folded structure, theoretically allowing cross-linking of engineered cysteines such as M348C and T1142C. Login to comment

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

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

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No. Sentence Comment
62 Fig. S2 demonstrates that the positively charged 2-trimethylaminoethyl MTS (MTSET+ ) adduct in TM12 enhances the effect of an anionic blocker, glibenclamide. Fig. S3 is a representative trace that illustrates protection of a substituted cysteine in TM12 by glibenclamide. Fig. S4 demonstrates modification of cysless/T1142C channels in the presence of ATP plus pyrophosphate. Login to comment
220 Because the modification rate of T1142C in Linsdell`s study is >60-fold faster than that of ours, we measured the modification rate of T1142C in the presence of ATP plus pyrophosphate and found that the modification rate is not significantly different from that measured with ATP alone (Fig. S4). Login to comment

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

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

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51 To investigate potential Cd2af9; bridges formed between pore-lining cysteine side chains exposed in the inner vestibule of the CFTR pore, we combined individual cysteines that we previously found to be accessible to cytoplasmically applied methanethiosulfonate reagents in three important pore-lining TMs: TM1 (K95C, Q98C) (13), TM6 (I344C, V345C, M348C, A349C) (15), and TM12 (M1140C, S1141C, T1142C, Q1144C, W1145C, V1147C, N1148C) (16), to generate a total of 50 double cysteine mutants (8 TM1:TM6; 14 TM1:TM12; 28 TM6:TM12). Login to comment
128 Also note that the estimated Ki values for some double mutants were c56;300 òe;M (V345C/M1140C, 316 afe; 38 òe;M, n afd; 3; A349C/M1140C, 345 afe; 58 òe;M, n afd; 3; A349C/T1142C, 231 afe; 68 òe;M, n afd; 3). Login to comment