ABCC7 p.Glu116Cys
| ClinVar: |
c.346G>A
,
p.Glu116Lys
?
, not provided
c.346G>C , p.Glu116Gln ? , not provided |
| CF databases: |
c.346G>A
,
p.Glu116Lys
(CFTR1)
D
, A missense mutation in exon 4 of the CFTR gene was detected by DGGE and identified by direct sequencing. The nucleotide position 478 is chnaged from G to A, leading to a subsitution of glutamic acid for lysine at position 116. This mutation abolishes a MnI restriction site. The mutation on the other chromosome is the deletion of [delta]F508
c.346G>C , p.Glu116Gln (CFTR1) ? , This mutation was detected in a thirty year old male with UAVD by multiplex heteroduplex analysis on the MDE gel matrix and direct sequencing. The patient was also heterozygous for the [delta]F508 mutation. Clinical Data: UAVD, borderline sweat test, chronic sinusitis |
| Predicted by SNAP2: | A: N (72%), C: D (59%), D: N (87%), F: D (71%), G: N (61%), H: D (63%), I: N (61%), K: N (61%), L: N (57%), M: D (63%), N: N (78%), P: N (57%), Q: N (82%), R: N (57%), S: N (78%), T: N (78%), V: N (66%), W: D (75%), Y: D (66%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, F: D, G: N, H: N, I: N, K: N, L: D, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: D, |
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[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
97 Opening rates for WTand R104C/ E116C-CFTR were measured as previously described except that R117A-CFTR were significantly lower than WT-CFTR (Fig. 2 D).
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ABCC7 p.Glu116Cys 25024266:97:31
status: NEW109 Therefore, we performed experiments to investigate the modification of WT-, D110C-, E116C-, and R117C-CFTR by MTSET (ET+ ) and MTSES (ES&#e032; ) with the TEVC technique; R334C-CFTR was used as a positive control (Zhang et al., 2005b).
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ABCC7 p.Glu116Cys 25024266:109:84
status: NEW140 However, under the same conditions, no functional modifications were observed for any of the three ECL1 cysteine mutants studied (D110C-, E116C-, and R117C-CFTR).
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ABCC7 p.Glu116Cys 25024266:140:138
status: NEW144 Our data could be interpreted in two ways: (1) the thiol groups of the engineered cysteines in D110C-, E116C-, and R117C-CFTR were not exposed and therefore unable to be modified by ET+ or ES&#e032; , or (2) the three cysteines Figure 2.ߓ Some ECL1 mutants exhibited decreased burst duration.
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ABCC7 p.Glu116Cys 25024266:144:103
status: NEW170 GlyH-101 blocked D110C- and R117C-CFTR similarly to WT-CFTR, whereas E116C-CFTR was also blocked significantly by GlyH-101 (P < 0.01), but less efficaciously than the other two mutants or the WT.
X
ABCC7 p.Glu116Cys 25024266:170:69
status: NEW189 The data presented so far resolve our first two questions in this paper: (1) Charge-swapping mutations of D110, E116, and R117 of ECL1 destabilize the open state, indicating that these residues contribute to maintaining the outer mouth open pore architecture of CFTR; (2) based Figure 4.ߓ Effects of 1 mM MTSET+ (ET+ ) and MTSES&#e032; (ES&#e032; ) on WT-, D110C-, E116C-, R117C-, and R334C-CFTR.
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ABCC7 p.Glu116Cys 25024266:189:371
status: NEW196 Figure 5.ߓ Effects of 2.5 &#b5;M GlyH-101 on WT-, D110C-, E116C-, and R117C-CFTR.
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ABCC7 p.Glu116Cys 25024266:196:64
status: NEW254 alone, R104C/E116C-CFTR exhibited very long stable openings with brief closed states and s1 and s2 subconductance states (Fig. 9 B, control).
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ABCC7 p.Glu116Cys 25024266:254:13
status: NEW260 If the long openings of R104C/E116C-CFTR were caused by the formation of a spontaneous disulfide bond, then the reducing agent DTT should break the disulfide bond and modify the channel behavior.
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ABCC7 p.Glu116Cys 25024266:260:30
status: NEW269 We reasoned that if a salt bridge between R104 and E116 is important for stabilizing the open state, the bifunctional linker MTS-2-MTS may lock the cysteine-substituted double mutant R104C/E116C-CFTR into the full open state by covalently binding to both engineered cysteines.
X
ABCC7 p.Glu116Cys 25024266:269:189
status: NEW283 To test this idea further, we preincubated oocytes expressing R104C/E116C-CFTR with 5 mM MTSET+ for Figure 9.ߓ E116 forms a salt bridge with R104 in both the closed and open states.
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ABCC7 p.Glu116Cys 25024266:283:68
status: NEW286 (B) Two cysteines engineered at positions 104 and 116 (R104C/E116C) form a spontaneous disulfide bond when CFTR is in the open state.
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ABCC7 p.Glu116Cys 25024266:286:61
status: NEW287 Representative single-channel trace of R104C/E116C-CFTR recorded with the same conditions as A.
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ABCC7 p.Glu116Cys 25024266:287:45
status: NEW290 +DTT in pipette: R104C/E116C-CFTR recorded with 1 mM DTT in the extracellular pipette solution (left, middle trace).
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ABCC7 p.Glu116Cys 25024266:290:23
status: NEW291 In the bottom trace, oocytes expressing R104C/ E116C-CFTR were incubated in solution containing 5 mM MTSET+ over 10 min before single-channel current recording (+MTSET; left, bottom trace).
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ABCC7 p.Glu116Cys 25024266:291:47
status: NEW293 Mean fraction of open burst duration is plotted at right for R104C/E116C-CFTR under three different experimental conditions, for each of the open conductance states: s1, dark red; s2, orange; and f, light green.
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ABCC7 p.Glu116Cys 25024266:293:67
status: NEW294 (C) Cross-linking R104C to E116C using MTS-2-MTS locks CFTR channels into the closed state.
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ABCC7 p.Glu116Cys 25024266:294:27
status: NEW295 Representative trace (left) and summary data (right) for macroscopic currents measured from R104C/E116C-CFTR with addition of 1 mM MTS-2-MTS in the absence of ISO at VM = &#e032;60 mV.
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ABCC7 p.Glu116Cys 25024266:295:98
status: NEW317 The required proximity for a salt bridge is confirmed by our finding that the thiol groups of two engineered cysteines at these positions are in close enough proximity in the open state to form a spontaneous disulfide bond (&#e07a;2-3 &#c5;); (b) R104C and/or E116C do not contribute directly to ion conduction and permeation through CFTR because both could be modified by MTSET without affecting channel conductance.
X
ABCC7 p.Glu116Cys 25024266:317:260
status: NEW318 In support of this, we found no detectable change in the macroscopic current of E116C-CFTR upon exposure to MTSET (Fig. 4), consistent with the notion that these ECL1 amino acids do not directly contribute to ion conduction and permeation in CFTR (Gao et al., 2013).
X
ABCC7 p.Glu116Cys 25024266:318:80
status: NEW319 The R104C/E116C spontaneous open state disulfide bond exhibited the following characteristics: (a) R104C/ E116C-CFTR still required ATP and PKA for activation.
X
ABCC7 p.Glu116Cys 25024266:319:10
status: NEWX
ABCC7 p.Glu116Cys 25024266:319:106
status: NEW320 (b) R104C/E116C-CFTR exhibited an intraburst closed state even in the absence of DTT that is long enough to represent true channel closures, suggesting that the spontaneous disulfide bond is not strong enough to lock the channel into the open state but rather the channel is still affected by NBD-mediated gating, although to a much lower degree than the WT.
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ABCC7 p.Glu116Cys 25024266:320:10
status: NEW323 Homology modeling and simulation predict that R104 and E116 might remain very close to each other and form a salt bridge when the channel is in the closed state as well; we therefore asked whether the bifunctional cross-linker MTS-2-MTS would lock R104C/E116C-CFTR closed when applied in the closed state.
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ABCC7 p.Glu116Cys 25024266:323:254
status: NEW324 Representative data are shown in Fig. 9 C. R104C/E116C-CFTR could be reversibly activated and reactivated by ISO (ISO1 and ISO2) without significant decrement.
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ABCC7 p.Glu116Cys 25024266:324:49
status: NEW328 The closed state MTS-2-MTS cross-link bond also showed a clear difference from the R104C/E116C open state spontaneous with a single exponential function with &#e074; = 5.33 min, which suggests that it takes &#e07a;7-8 min for WT-CFTR current to reach its plateau.
X
ABCC7 p.Glu116Cys 25024266:328:89
status: NEW422 We found that E116 of ECL1 forms a salt bridge with R104 of TM1 in both the closed and open states, and the two amino acids are very close to each other when the channel is in the open state because R104C only forms a spontaneous disulfide bond with E116C in this state.
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ABCC7 p.Glu116Cys 25024266:422:250
status: NEW430 To further test the existence of a possible open state salt bridge between R117 and E1126, we again asked whether cysteines engineered at positions 117 and 1126 might form a disulfide bond as in R104C/E116C- or D110C/K892C-CFTR.
X
ABCC7 p.Glu116Cys 25024266:430:201
status: NEW493 In the current study, we identified two spontaneous disulfide bonds in CFTR formed after introduction of cysteines at positions 110 and 892 (a closed state disulfide bond in D110C/K892C-CFTR) or 104 and 116 (an open state disulfide bond in R104C/E116C-CFTR).
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ABCC7 p.Glu116Cys 25024266:493:246
status: NEW496 In contrast, the R104C/E116C disulfide bond was occasionally broken during normal NBD-mediated gating in the presence of ATP, although the closed state was rather brief.
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ABCC7 p.Glu116Cys 25024266:496:23
status: NEW498 In contrast, R104C/E116C forms a relatively weak disulfide bond, most likely because of its dihedral angle being dramatically off from 90&#b0;, and thus the dissociation energy is likely below that of ATP hydrolysis and subsequent NBD dedimerization.
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ABCC7 p.Glu116Cys 25024266:498:19
status: NEW