ABCC7 p.Met348Cys
| ClinVar: |
c.1043T>A
,
p.Met348Lys
?
, not provided
|
| CF databases: |
c.1042A>G
,
p.Met348Val
(CFTR1)
?
, This mutation was identified in a CFTR gene mutation screening of 60 Patients with idiopathic chronic pancreatitis recruited from the region of North Rhine Westfalia in Germany. The entire coding region of the CFTR gene was sequenced.
c.1043T>A , p.Met348Lys (CFTR1) ? , The mutation on the other chromosome is still unknown. This mutation was found on one chromosome while screening 56 Italian CF chromosomes. c.1043T>C , p.Met348Thr (CFTR1) ? , The mutation was detected by DGGE analysis and characterized by direct sequencing. We have seen it only twice, in over 1800 control chromosomes from Italian population. |
| Predicted by SNAP2: | A: D (71%), C: D (66%), D: D (85%), E: D (85%), F: D (63%), G: D (80%), H: D (80%), I: N (53%), K: D (85%), L: N (66%), N: D (71%), P: D (91%), Q: D (63%), R: D (85%), S: D (66%), T: D (75%), V: N (53%), W: D (80%), Y: D (75%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: N, G: D, H: D, I: N, K: D, L: N, N: D, P: D, Q: D, R: D, S: D, T: D, 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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No. Sentence Comment
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.
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ABCC7 p.Met348Cys 15272010:56:21
status: NEW146 Three positive cross-linking mutants, M348C/T1142C, T351C/T1142C, and W356C/W1145C were identified (see Fig. 3B, band X) and selected for further study.
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ABCC7 p.Met348Cys 15272010:146:38
status: NEW148 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.
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ABCC7 p.Met348Cys 15272010:148:69
status: NEWX
ABCC7 p.Met348Cys 15272010:148:142
status: NEW150 The cross-linking patterns of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C showed differences when treated with different cross-linkers.
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ABCC7 p.Met348Cys 15272010:150:38
status: NEW151 Mutant M348C/T1142C, for example, showed cross-linking with M5M and M8M but not with M17M.
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ABCC7 p.Met348Cys 15272010:151:7
status: NEW153 It is interesting to note that both M348C and T351C in TM6 showed cross-linking to T1142C in TM12.
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ABCC7 p.Met348Cys 15272010:153:36
status: NEW159 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.
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ABCC7 p.Met348Cys 15272010:159:35
status: NEWX
ABCC7 p.Met348Cys 15272010:159:176
status: NEW182 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).
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ABCC7 p.Met348Cys 15272010:182:186
status: NEW183 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.
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ABCC7 p.Met348Cys 15272010:183:116
status: NEW187 Each cDNA contained one of the cysteine mutations M348C, T351C, W356C, T1142C, or W1145C.
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ABCC7 p.Met348Cys 15272010:187:50
status: NEW188 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).
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ABCC7 p.Met348Cys 15272010:188:63
status: NEW189 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.
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ABCC7 p.Met348Cys 15272010:189:257
status: NEW191 As shown in Fig. 6A, incorporation of the ⌬F508 mutation into mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C abolished cross-linking.
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ABCC7 p.Met348Cys 15272010:191:77
status: NEW196 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.
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ABCC7 p.Met348Cys 15272010:196:201
status: NEW197 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.
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ABCC7 p.Met348Cys 15272010:197:96
status: NEW212 As shown in Fig. 6B, brefeldin A blocked processing of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C.
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ABCC7 p.Met348Cys 15272010:212:63
status: NEW215 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.
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ABCC7 p.Met348Cys 15272010:215:35
status: NEW235 The M348C/T1142C mutant showed a similar level of activity as WT CFTR.
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ABCC7 p.Met348Cys 15272010:235:4
status: NEW248 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."
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ABCC7 p.Met348Cys 15272010:248:137
status: NEW262 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.
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ABCC7 p.Met348Cys 15272010:262:40
status: NEW263 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.
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ABCC7 p.Met348Cys 15272010:263:66
status: NEW266 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.
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ABCC7 p.Met348Cys 15272010:266:70
status: NEW268 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.
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ABCC7 p.Met348Cys 15272010:268:96
status: NEW[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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No. Sentence Comment
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.
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ABCC7 p.Met348Cys 18056267:100:312
status: NEW[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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No. Sentence Comment
230 An example is the channel blocker, benzbromarone, which also inhibited cross-linking between cysteine residues in TM6 and TM7 [I340C(TM6)/S877C(TM7) mutant] [17], as well as between cysteine residues in TM6 and TM12 [M348C- (TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants] (Figure 4).
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ABCC7 p.Met348Cys 18361776:230:217
status: NEW[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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No. Sentence Comment
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).
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ABCC7 p.Met348Cys 18597042:153:100
status: NEW[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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No. Sentence Comment
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.
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ABCC7 p.Met348Cys 19754156:52:806
status: NEW[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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No. Sentence Comment
17 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.
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ABCC7 p.Met348Cys 20805575:17:14
status: NEW107 Spontaneous ATP-independent gating of cysless/I344C and cysless/M348C was also increased by MTSET because after the removal of ATP, there remained a substantial amount of current, which can be inhibited by CFTR-specific inhibitor, K335C, F337, and T338C at 50 mV membrane potential (0.46 pA for cysless/WT).
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ABCC7 p.Met348Cys 20805575:107:64
status: NEW120 In addition, MTSET modification can more than double the current in M348C channels.
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ABCC7 p.Met348Cys 20805575:120:68
status: NEW123 A representative result of the experiments with the cysless/ M348C construct is shown in Fig. 5.
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ABCC7 p.Met348Cys 20805575:123:61
status: NEW135 (D) MTSET increased ATP-dependent current in cysless/M348C channels.
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ABCC7 p.Met348Cys 20805575:135:53
status: NEW146 Interestingly, MTSET modification of M348C also slightly but significantly increased (12 ± 1%; n = 5) the single-channel amplitude (Fig. 5, A and C).
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ABCC7 p.Met348Cys 20805575:146:37
status: NEW154 Again, gating Figure 5. Effects of MTSET on a single cysless/M348C channel.
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ABCC7 p.Met348Cys 20805575:154:69
status: NEW156 (B) Gating parameters of the cysless/M348C channel before (black) and after (blue) modification, as extracted from the traces in A. Those of the cysless/ WT (gray) in the presence of 2 mM ATP are also included for comparison (traces not depicted).
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ABCC7 p.Met348Cys 20805575:156:37
status: NEW157 n = 5 for cysless/M348C.
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ABCC7 p.Met348Cys 20805575:157:18
status: NEW159 (C) Single-channel amplitude of the cysless/M348C channel before and after modification in the same patch.
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ABCC7 p.Met348Cys 20805575:159:44
status: NEW186 Instead, we will focus on the four other positive hits (i.e., I344C, V345C, M348C, and Q353C).
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ABCC7 p.Met348Cys 20805575:186:76
status: NEW201 (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.
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ABCC7 p.Met348Cys 20805575:201:306
status: NEW212 Similar results were obtained with the cysless/V345C and cysless/ M348C channels.
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ABCC7 p.Met348Cys 20805575:212:66
status: NEW256 (A and B) Macroscopic recordings of cysless/ V345C and cysless/M348C showing modification by 1 mM MTSES when the membrane potential is held at 50 mV (left) and 100 mV (right).
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ABCC7 p.Met348Cys 20805575:256:63
status: NEW279 Similar results were obtained for cysless/M348C: 0.36 ± 0.03 s (n = 3) before and 0.55 ± 0.03 s (n = 3) after modification.
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ABCC7 p.Met348Cys 20805575:279:42
status: NEW290 For example, biochemical studies demonstrated that both M348C and T351C can be cross-linked to T1142C in TM12 (Chen et al. 2004).
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ABCC7 p.Met348Cys 20805575:290:56
status: NEW316 In fact, our data show that the Po of spontaneous gating in the absence of ATP (Bompadre et al., 2007; Wang et al., 2010) is visibly increased by MTSET modification (Figs. 4 D and 12 B; Po is 0.19 ± 0.04, n = 5 for cysless/M348C, and 0.63 ± 0.03, n = 4 for cysless/ I344C).
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ABCC7 p.Met348Cys 20805575:316:228
status: NEW[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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No. Sentence Comment
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).
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ABCC7 p.Met348Cys 21520952:28:185
status: NEW50 (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.
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ABCC7 p.Met348Cys 21520952:50:90
status: NEW[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.
Comments [show]
None has been submitted yet.
No. Sentence 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].
X
ABCC7 p.Met348Cys 21796338:140:339
status: NEW207 However, charge-conservative mutations in the analgous part of TM6-for example, in I344C, V345C, M348C, and A349C-also failed to significantly alter Cl-conductance [4].
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ABCC7 p.Met348Cys 21796338:207:97
status: NEW[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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
259 Chen et al.41 observed cross-linking of M348C and T1142C by M5M and M8M but not M17M.
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ABCC7 p.Met348Cys 22352759:259:40
status: NEW262 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.
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ABCC7 p.Met348Cys 22352759:262:199
status: NEW[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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None has been submitted yet.
No. Sentence Comment
198 (C) Second-order rate constants (MTSES ) of Texas red MTSEA+ modification for cysless/ S1141C-, cysless/N1148C-, cysless/ I344C-, and cysless/M348C-CFTR channels.
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ABCC7 p.Met348Cys 22042986:198:159
status: NEW[hide] Alignment of transmembrane regions in the cystic f... J Gen Physiol. 2011 Aug;138(2):165-78. Epub 2011 Jul 11. Wang W, El Hiani Y, Linsdell P
Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Gen Physiol. 2011 Aug;138(2):165-78. Epub 2011 Jul 11., [PMID:21746847]
Abstract [show]
Different transmembrane (TM) alpha helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl(-) channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84-T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.
Comments [show]
None has been submitted yet.
No. Sentence Comment
139 Our work concerning intracellular MTS reagent modification in TM6 also identified some cysteines that could be modified in both activated and nonactivated channels (e.g., V345C and M348C), and others that could apparently be modified only after channel activation (e.g., T338C, S341C, and I344C), suggesting a state-dependent conformational change that alters access of internally applied MTS reagents into the pore (El Hiani and Linsdell, 2010).
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ABCC7 p.Met348Cys 21746847:139:181
status: NEW162 (A-C) Example leak-subtracted I-V relationships for K95C/I344C (A), Q98C/I344C (B), and Q98C/M348C (C) after channel activation with 20 nM PKA and 1 mM ATP.
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ABCC7 p.Met348Cys 21746847:162:93
status: NEW166 Note that cys-less CFTR, the single mutants K95C, Q98C, or I344C, and the double mutant Q98C/M348C were all insensitive to CuPhe under these conditions.
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ABCC7 p.Met348Cys 21746847:166:93
status: NEW182 However, K95C/ I344C, Q98C/I344C, and Q98C/M348C did generate macroscopic PKA- and ATP-dependent currents in inside-out patches.
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ABCC7 p.Met348Cys 21746847:182:43
status: NEW186 Furthermore, the lack of effect of CuPhe on Q98C/M348C indicated that not all double-cysteine mutants were CuPhe sensitive, which we take to indicate that only nearby cysteine side chains can be cross-linked by this reagent.
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ABCC7 p.Met348Cys 21746847:186:49
status: NEW244 In contrast, we found no evidence for disulfide bond formation between a pair of introduced cysteine side chains that would be predicted (based on Fig. 9) to be further apart, Q98C in TM1 and M348C in TM6 (Fig. 6, C and D).
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ABCC7 p.Met348Cys 21746847:244:192
status: NEW256 For comparison, the MTSES modification rate constant for P99C and L102C (Fig. 3) was similar to that of T338C and S341C in TM6 (El Hiani and Linsdell, 2010) (all between 100 and 150 M1 s1 ), and the modification rate constant for K95C was comparable to, or slightly greater than, that of I344C, V345C, and M348C (El Hiani and Linsdell, 2010) (all between 2,000 and 4,000 M1 s1 ).
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ABCC7 p.Met348Cys 21746847:256:322
status: NEW[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.
Comments [show]
None has been submitted yet.
No. Sentence 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.
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ABCC7 p.Met348Cys 8744306:91:422
status: NEW90 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.
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ABCC7 p.Met348Cys 8744306:90:422
status: NEW[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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
185 Likewise in Bai et al. (8), chemical modifications of an engineered cysteine (I344C or M348C) in TM6 drastically increase ATP-independent activity to the level of ATP-dependent activity before modifications.
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ABCC7 p.Met348Cys 23223629:185:87
status: NEW[hide] Cysteine scanning of CFTR's first transmembrane se... Biophys J. 2013 Feb 19;104(4):786-97. doi: 10.1016/j.bpj.2012.12.048. Gao X, Bai Y, Hwang TC
Cysteine scanning of CFTR's first transmembrane segment reveals its plausible roles in gating and permeation.
Biophys J. 2013 Feb 19;104(4):786-97. doi: 10.1016/j.bpj.2012.12.048., [PMID:23442957]
Abstract [show]
Previous cysteine scanning studies of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have identified several transmembrane segments (TMs), including TM1, 3, 6, 9, and 12, as structural components of the pore. Some of these TMs such as TM6 and 12 may also be involved in gating conformational changes. However, recent results on TM1 seem puzzling in that the observed reactive pattern was quite different from those seen with TM6 and 12. In addition, whether TM1 also plays a role in gating motions remains largely unknown. Here, we investigated CFTR's TM1 by applying methanethiosulfonate (MTS) reagents from both cytoplasmic and extracellular sides of the membrane. Our experiments identified four positive positions, E92, K95, Q98, and L102, when the negatively charged MTSES was applied from the cytoplasmic side. Intriguingly, these four residues reside in the extracellular half of TM1 in previously defined CFTR topology; we thus extended our scanning to residues located extracellularly to L102. We found that cysteines introduced into positions 106, 107, and 109 indeed react with extracellularly applied MTS probes, but not to intracellularly applied reagents. Interestingly, whole-cell A107C-CFTR currents were very sensitive to changes of bath pH as if the introduced cysteine assumes an altered pKa-like T338C in TM6. These findings lead us to propose a revised topology for CFTR's TM1 that spans at least from E92 to Y109. Additionally, side-dependent modifications of these positions indicate a narrow region (L102-I106) that prevents MTS reagents from penetrating the pore, a picture similar to what has been reported for TM6. Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR's gating cycle. The structural implications of these findings are discussed in light of the crystal structures of ABC transporters and homology models of CFTR.
Comments [show]
None has been submitted yet.
No. Sentence Comment
164 Similar to what we observed for I344C- and M348C-CFTR (16), this robust ATP-independent gating was seen following modification by MTSET but not by MTS-ethylammonium (MTSEA) (Fig. S4).
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ABCC7 p.Met348Cys 23442957:164:43
status: NEW[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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
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).
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ABCC7 p.Met348Cys 25143385:51:351
status: NEW71 In contrast, the remaining seven double cysteine mutants, namely I344C/S1141C (Fig. 2, C and D), V345C/S1141C, M348C/ S1141C (Fig. 2, C and E), M348C/V1144C, M348C/W1145C, M348C/V1147C, and M348C/N1148C, all showed increased sensitivity to Cd2af9; , leading to a significant decrease in Ki as compared with either of the single cysteine mutants from which they were derived (estimated Ki values b0d; 50 òe;M; Fig. 3).
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ABCC7 p.Met348Cys 25143385:71:111
status: NEWX
ABCC7 p.Met348Cys 25143385:71:144
status: NEWX
ABCC7 p.Met348Cys 25143385:71:158
status: NEWX
ABCC7 p.Met348Cys 25143385:71:172
status: NEWX
ABCC7 p.Met348Cys 25143385:71:190
status: NEW77 In particular, M348C/S1141C was associated with a dramatic increase in sensitivity to Cd2af9; (Fig. 2, C and E, and Fig. 3) with an estimated Ki of 0.14 afe; 0.02 òe;M (n afd; 5).
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ABCC7 p.Met348Cys 25143385:77:15
status: NEW80 In each case, PPi treatment resulted in a weakening of Cd2af9; inhibition (Fig. 4A) and a significant increase in Ki (Fig. 4B) of between 2.3-fold (in I344C/S1141C) and 97-fold (in M348C/ S1141C).
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ABCC7 p.Met348Cys 25143385:80:184
status: NEW83 As shown in Fig. 5, all E1371Q-containing channels tested were only weakly sensitive to inhibition by Cd2af9; , resulting in a significant increase in Ki both in single cysteine (I344C, M348C, S1141C) and in double cysteine (I344C/S1141C, Fig. 5, A-C; M348C/S1141C, Fig. 5, A, D, and E) mutant channels.
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ABCC7 p.Met348Cys 25143385:83:189
status: NEWX
ABCC7 p.Met348Cys 25143385:83:255
status: NEW84 However, the effect of the E1371Q mutation was greater in the double cysteine mutants; this gating mutation increased Ki 30-fold in I344C/S1141C (Fig. 5C) and 2500-fold in M348C/S1141C (Fig. 5E).
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ABCC7 p.Met348Cys 25143385:84:172
status: NEW98 C, sample time courses (upper panels) and I-V curves (lower panels) recorded from similar experiments for the double cysteine mutants I344C/S1141C (left) and M348C/S1141C (right).
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ABCC7 p.Met348Cys 25143385:98:158
status: NEW127 Note that the Ki for M348C/S1141C (0.14 afe; 0.02 òe;M, n afd; 5) is too small to be visible on this scale, but was significantly different from either M348C or S1141C alone (p b0d; 0.0005; see also Fig. 2E).
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ABCC7 p.Met348Cys 25143385:127:21
status: NEWX
ABCC7 p.Met348Cys 25143385:127:162
status: NEW137 Thus, M348C is able to form Cd2af9; bridges with cysteines at multiple positions in TM12 (S1141C, Q1144C, W1145C, V1147C, N1148C) (Fig. 8B), and S1141C is able to form Cd2af9; bridges with cysteines both in TM1 (K95C) and in TM6 (I344C, V345C, M348C) (Fig. 8C).
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ABCC7 p.Met348Cys 25143385:137:6
status: NEWX
ABCC7 p.Met348Cys 25143385:137:250
status: NEW139 For example, M348C is able to form Cd2af9; bridges with TM12 sites on a broad face of the TM12 helical face (Fig. 8B, top panel), suggesting some degree of relative rotational flexibility or movement, and also across a distance spanning two helical turns (from Ser-1141 to Asn-1148) (Fig. 8B, bottom panel), suggesting also some translational flexibility or movement.
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ABCC7 p.Met348Cys 25143385:139:13
status: NEW146 Most striking in this respect was the M348C/S1141C mutant FIGURE 4.
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ABCC7 p.Met348Cys 25143385:146:38
status: NEW148 A, mean fractional current remaining following the addition of different concentrations of Cd2af9; in M348C/S1141C channels in the presence of PKA and ATP (F) or following activation by PKA and ATP followed by treatment with 2 mM PPi to maximize channel open probability (E).
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ABCC7 p.Met348Cys 25143385:148:105
status: NEW152 The unusually high apparent Cd2af9; binding affinity of this double cysteine mutant (Figs. 3B and 4B), more than 700-fold lower Ki than the corresponding single cysteine mutants (Figs. 2E and 3), suggests that M348C and S1141C are uniquely well positioned to coordinate tight Cd2af9; binding in closed channels.
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ABCC7 p.Met348Cys 25143385:152:213
status: NEW153 However, when the channel is open, these two cysteines do not appear to coordinate Cd2af9; at all because the apparent Cd2af9; affinity in M348C/S1141C/ E1371Q channels appears the same as in M348C/E1371Q or S1141C/E1371Q (Fig. 5, D and E).
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ABCC7 p.Met348Cys 25143385:153:145
status: NEWX
ABCC7 p.Met348Cys 25143385:153:198
status: NEW164 A, sample time courses and I-V curves illustrating the low Cd2af9; sensitivity of constitutively active I344C/S1141C/E1371Q (left panels) and M348C/S1141C/E1371Q (right panels) channels in inside-out patches. Experiments were performed as described in the legend for Fig. 2.
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ABCC7 p.Met348Cys 25143385:164:145
status: NEW175 As described above, several different Cd2af9; bridges can form between M348C (TM6) and TM12 (Fig. 8, A and B), as well as between S1141C (TM12) and TM6; all appear to stabilize the closed state (Figs. 3 and 4).
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ABCC7 p.Met348Cys 25143385:175:74
status: NEW195 )Residuesinredarethosewithpore-liningsidechainsmutatedinthepresentstudy;thoseinblackarenon-pore-lining.Redlinesconnectresiduesthat canformCd2af9; bridgesfollowingmutationtocysteine.B,relativelocationandorientationofMet-348(TM6)withthoseresiduesinTM12,whichcanformCd2af9; bridges with M348C following mutation to cysteine.
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ABCC7 p.Met348Cys 25143385:195:290
status: NEW[hide] Timing of CFTR Pore Opening and Structure of Its T... Cell. 2015 Oct 22;163(3):724-33. doi: 10.1016/j.cell.2015.09.052. Epub 2015 Oct 22. Sorum B, Czege D, Csanady L
Timing of CFTR Pore Opening and Structure of Its Transition State.
Cell. 2015 Oct 22;163(3):724-33. doi: 10.1016/j.cell.2015.09.052. Epub 2015 Oct 22., [PMID:26496611]
Abstract [show]
In CFTR, the chloride ion channel mutated in cystic fibrosis (CF) patients, pore opening is coupled to ATP-binding-induced dimerization of two cytosolic nucleotide binding domains (NBDs) and closure to dimer disruption following ATP hydrolysis. CFTR opening rate, unusually slow because of its high-energy transition state, is further slowed by CF mutation DeltaF508. Here, we exploit equilibrium gating of hydrolysis-deficient CFTR mutant D1370N and apply rate-equilibrium free-energy relationship analysis to estimate relative timing of opening movements in distinct protein regions. We find clear directionality of motion along the longitudinal protein axis and identify an opening transition-state structure with the NBD dimer formed but the pore still closed. Thus, strain at the NBD/pore-domain interface, the DeltaF508 mutation locus, underlies the energetic barrier for opening. Our findings suggest a therapeutic opportunity to stabilize this transition-state structure pharmacologically in DeltaF508-CFTR to correct its opening defect, an essential step toward restoring CFTR function.
Comments [show]
None has been submitted yet.
No. Sentence Comment
74 Timing of Motion at Position 348 in the Pore Region (A) Inward single-channel currents of the cut-DR(D1370N) CFTR background construct (top trace) and of channels bearing mutations M348I, M348K, M348C, M348N, and M348A, respectively, in the same background. Currents were recorded at 80 mV, in symmetrical 140 mM Cl ; dashes on the left mark zero-current level.
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ABCC7 p.Met348Cys 26496611:74:195
status: NEW