ABCC7 p.Asp993Cys
| ClinVar: |
c.2977G>T
,
p.Asp993Tyr
?
, not provided
|
| CF databases: |
c.2977G>T
,
p.Asp993Tyr
(CFTR1)
D
, The above mutation was found by DGGE and then direct sequencing of DNA from a patient with severe phenotype from Southern France.
c.2978A>G , p.Asp993Gly (CFTR1) ? , The mutation was detected by DHPLC analysis and characterized by direct sequencing |
| Predicted by SNAP2: | A: D (71%), C: D (71%), E: D (66%), F: D (80%), G: D (80%), H: D (80%), I: D (75%), K: D (91%), L: D (85%), M: D (75%), N: D (80%), P: D (91%), Q: D (80%), R: D (91%), S: D (71%), T: D (80%), V: D (80%), W: D (85%), Y: D (85%), |
| Predicted by PROVEAN: | A: N, C: D, E: N, F: D, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: D, |
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[hide] Two salt bridges differentially contribute to the ... J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24. Cui G, Freeman CS, Knotts T, Prince CZ, Kuang C, McCarty NA
Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function.
J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24., [PMID:23709221]
Abstract [show]
Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.
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No. Sentence Comment
72 Open burst durations and closed durations were measured from single channel recordings of WT-, R352C-, D993C-, and R352C/D993C-CFTR, and then histograms and fits of them with single exponential functions were generated with IGOR (WaveMetrics, Inc., Lake Oswego, OR) to determine time constants for open burst durations (o, also called beta) and closed durations (c, also called ॷ) for all of the constructs.
X
ABCC7 p.Asp993Cys 23709221:72:103
status: NEWX
ABCC7 p.Asp993Cys 23709221:72:121
status: NEW152 We generated R352C/D993C-CFTR and exposed the channels to MTS-2-MTS; MTS-2-MTS was chosen for this experiment because it leads to cross-linking of cysteines at a distance of b03;4.6 &#c5;, which is within the average distance for known salt bridges in a varietyofproteins(13).Priortostudieswiththedoublemutant,we also investigated each single mutant and their responses to monofunctional sulfhydryl-modifying reagents.
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ABCC7 p.Asp993Cys 23709221:152:19
status: NEW162 Recovery of Charge at R352C and D993C Rescued Channel Stability in the Full Open State-R352C-CFTR exhibited single channel behavior similar to that previously reported for R352A-, R352Q-, and R352E-CFTR (13).
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ABCC7 p.Asp993Cys 23709221:162:32
status: NEW176 In contrast to these results for R352C-CFTR, the stability of single channel opening in D993C-CFTR was rescued to mimic that of WT-CFTR by exposure to MTSESafa; (but not MTSEAaf9; or MTSETaf9; ), leading to significantly increased mean burst duration (supplemental Fig. 3B).
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ABCC7 p.Asp993Cys 23709221:176:88
status: NEW177 Exposure to MTSESafa; also significantly decreased the conductance of the f state of D993C-CFTR (supplemental Fig. 3B).
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ABCC7 p.Asp993Cys 23709221:177:88
status: NEW179 We repeated the above experiments in R352C/Cys-less V510A-CFTR and D993C/ Cys-less V510A-CFTR to further rule out the possibility of any endogenous cysteines being involved in the process.
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ABCC7 p.Asp993Cys 23709221:179:67
status: NEW182 A Bifunctional MTS Reagent Can Latch R352C/D993C-CFTR into the Full Open State Even after Washout of ATP-We hypothesized that the CFTR channel pore could be latched into the open state by cross-linking the two cysteines at R352C and D993C.
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ABCC7 p.Asp993Cys 23709221:182:43
status: NEWX
ABCC7 p.Asp993Cys 23709221:182:233
status: NEW183 We first tested the effects of monofunctional reagents MTSETaf9; , MTSEAaf9; , and MTSESafa; on the double mutant R352C/D993C-CFTR.
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ABCC7 p.Asp993Cys 23709221:183:129
status: NEW184 None of these reagents rescued salt Dynamic Modulation of the CFTR Pore by Salt Bridges JULY 12, 2013ߦVOLUME 288ߦNUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20763 bridge function to stabilize channel behavior in R352C/D993C-CFTR in terms of stable openings to the f state (data not shown).
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ABCC7 p.Asp993Cys 23709221:184:229
status: NEW185 Before applying MTS-2-MTS to the R352C/D993C-CFTR double mutant, we first tested the effects of this bifunctional linker on WT-CFTR (Fig. 6A) and Cys-less V510A-CFTR (Fig. 6B).
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ABCC7 p.Asp993Cys 23709221:185:39
status: NEW187 We then examined the effects of MTS-2-MTS on R352C-D993C-CFTR (on the WT-CFTR background); a representative experiment is shown in Fig. 7.
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ABCC7 p.Asp993Cys 23709221:187:51
status: NEW188 In the presence of ATP and PKA, prior to the addition of MTS-2-MTS, R352C/D993C-CFTR exhibited low open probability, unstable openings to the f state, and occasional subconductance open states.
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ABCC7 p.Asp993Cys 23709221:188:74
status: NEW191 It seems likely that this reflects the fact that there are several possible consequences of exposing the double mutant to MTS-2-MTS, including covalent modification of R352C and D993C separately by two MTS-2-MTS molecules within each CFTR protein.
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ABCC7 p.Asp993Cys 23709221:191:178
status: NEW209 The free energy change èc;èc;G between WT-CFTR and R352C/D993C-CFTR was afa;1.508 kcal/mol, which suggests that R352C and D993C interact with each other when CFTR is in the open state (supplemental Fig. 5).
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ABCC7 p.Asp993Cys 23709221:209:65
status: NEWX
ABCC7 p.Asp993Cys 23709221:209:133
status: NEW218 A, effects of 100 òe;M MTS-2-MTS on R352C-D993C-CFTR.
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ABCC7 p.Asp993Cys 23709221:218:46
status: NEW220 B, MTS-2-MTS failed to functionally modify R352C/D993C-CFTR when applied when the channel was in the closed state.
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ABCC7 p.Asp993Cys 23709221:220:49
status: NEW[hide] Three charged amino acids in extracellular loop 1 ... J Gen Physiol. 2014 Aug;144(2):159-79. doi: 10.1085/jgp.201311122. Epub 2014 Jul 14. Cui G, Rahman KS, Infield DT, Kuang C, Prince CZ, McCarty NA
Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR.
J Gen Physiol. 2014 Aug;144(2):159-79. doi: 10.1085/jgp.201311122. Epub 2014 Jul 14., [PMID:25024266]
Abstract [show]
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1-6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5'-(beta,gamma-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues.
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No. Sentence Comment
268 To further test the possible salt bridge between R104 and E116, we made use of MTS reagents that we used previously to confirm interactions between R352C and D993C (Cui et al., 2013).
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ABCC7 p.Asp993Cys 25024266:268:158
status: NEW321 This is in contrast to the ability of MTS-2-MTS to lock R352C/D993C-CFTR into the open state or to lock R334C/E217C-CFTR into the closed state (Cui et al., 2013; Rahman et al., 2013).
X
ABCC7 p.Asp993Cys 25024266:321:62
status: NEW