ABCC7 p.Lys892Cys
| Predicted by SNAP2: | A: N (72%), C: D (53%), D: N (61%), E: N (82%), F: D (63%), G: N (66%), H: N (78%), I: N (66%), L: N (72%), M: N (72%), N: N (78%), P: N (53%), Q: N (82%), R: N (82%), S: N (82%), T: N (82%), V: N (66%), W: D (66%), Y: D (59%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: N, Y: N, |
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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
93 Window current (calculated as the rolling mean of current in a 1-min window, in pA) over each minute for WTand D110C/K892C-CFTR records was measured sequentially for 21 min with Clampfit 10.2.
X
ABCC7 p.Lys892Cys 25024266:93:117
status: NEW119 Fig. S7 shows representative TEVC current traces of WTand D110C/ K892C-CFTR with DTT pretreatment.
X
ABCC7 p.Lys892Cys 25024266:119:65
status: NEW310 To test this, we made the double cysteine mutant D110C/K892C-CFTR.
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ABCC7 p.Lys892Cys 25024266:310:55
status: NEW312 The data also suggested that at least some of the double mutant D110C/K892C-CFTR channels could be activated by ATP and PKA in the absence of DTT, which would not support the formation of a stable spontaneous disulfide bond.
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ABCC7 p.Lys892Cys 25024266:312:70
status: NEW313 To resolve this, we backfilled 1 mM DTT in the pipette solution and recorded single-channel current of WTand D110C/K892C-CFTR from inside-out patches in the presence of cytosolic ATP and PKA, whereas DTT diffused to the tip.
X
ABCC7 p.Lys892Cys 25024266:313:115
status: NEW329 D110C/K892C-CFTR patch current remained low in the first &#e07a;5 min, then slowly increased over the next 10-15 min as more channels were activated and the number of apparent channels in the patch increased (Fig. 10 C).
X
ABCC7 p.Lys892Cys 25024266:329:6
status: NEW332 However, patches from oocytes expressing D110C/K892C-CFTR exhibited a very large increase in apparent channel number after exposure to extracellular DTT, consistent with channels being released from the spontaneous disulfide bond and therefore able to open.
X
ABCC7 p.Lys892Cys 25024266:332:47
status: NEW335 (A) Representative single-channel current traces of K892E-, D110R/K892E-, and D110C/K892C-CFTR recorded with the same experimental conditions as Fig. 2 (left), their all-points amplitude histograms (middle), and their mean burst durations (right).
X
ABCC7 p.Lys892Cys 25024266:335:84
status: NEW336 *, P < 0.05 indicates a significant difference between D110R- and D110R/K892E-CFTR; #, P < 0.001 for D110C/K892C-CFTR compared with D110R alone.
X
ABCC7 p.Lys892Cys 25024266:336:107
status: NEW342 (C) Exposure to 1 mM DTT, backfilled into the pipette, increased the number of active channels in D110C/K892C-CFTR.
X
ABCC7 p.Lys892Cys 25024266:342:104
status: NEW347 for several minutes, the question remained as to whether D110C/K892C-CFTR channels form a spontaneous disulfide bond in intact cells where CFTR is in the constant presence of ATP.
X
ABCC7 p.Lys892Cys 25024266:347:63
status: NEW350 In contrast, in D110C/K892C-CFTR, activation by ISO plus DTT (ISO2 + DTT) led to significantly higher current than ISO alone (ISO1).
X
ABCC7 p.Lys892Cys 25024266:350:22
status: NEW351 These data suggest that in whole oocytes, a fraction of D110C/ K892C channels formed disulfide bonds under resting conditions and were locked into the closed state (C0).
X
ABCC7 p.Lys892Cys 25024266:351:63
status: NEW353 We then asked whether DTT could further activate D110C/K892C-CFTR current if we used DTT before ISO.
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ABCC7 p.Lys892Cys 25024266:353:55
status: NEW354 We used 1 mM DTT alone for 3 min followed by ISO alone (ISO1), which fully activated D110C/K892C-CFTR channels to plateau.
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ABCC7 p.Lys892Cys 25024266:354:91
status: NEW357 These data suggest that with prior DTT treatment, disulfide bonds formed during the closed state in D110C/K892C-CFTR were broken by DTT and ISO1 was able to activate all channels to reach maximum current.
X
ABCC7 p.Lys892Cys 25024266:357:106
status: NEW361 In the absence of 1 mM DTT, D110C/K892C-CFTR channels were activated by ISO1 and ISO2 to a similar level (Fig. S7 C), suggesting that ISO alone was not able to break the spontaneous disulfide bond between K892C and D110C in channels that had formed this bond.
X
ABCC7 p.Lys892Cys 25024266:361:34
status: NEWX
ABCC7 p.Lys892Cys 25024266:361:205
status: NEW362 In summary, the above data, combined with molecular modeling, suggest three important findings: (1) D110 forms a salt bridge with K892 in the closed state; (2) D110C/K892C-CFTR forms a spontaneous disulfide bond when the channel is in the closed state and the energy of CFTR channel gating is not strong enough to break it in the absence of the reducing agent DTT; (3) CFTR may transition to a state where the NBDs are fully dedimerized (C0 closed state), as our simulations suggest that C0 is the only state where these residues approach each other closely enough for a spontaneous disulfide to form.
X
ABCC7 p.Lys892Cys 25024266:362:166
status: NEW363 unlike WT-CFTR, D110C/K892C-CFTR was modified by DTT when it perfused to the pipette tip over 5-10 min during channel phosphorylation; DTT probably broke the disulfide bond between D110C and K892C, allowing more channels to be activated by ATP and PKA.
X
ABCC7 p.Lys892Cys 25024266:363:22
status: NEWX
ABCC7 p.Lys892Cys 25024266:363:191
status: NEW364 The single-channel current amplitude of D110C/K892C-CFTR remained unchanged in the absence and presence of DTT; individual single-channel openings in the presence of DTT could not be distinguished from those in channels that were able to open before DTT.
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ABCC7 p.Lys892Cys 25024266:364:46
status: NEW367 Collectively, the data suggest that D110C/ K892C-CFTR forms a spontaneous disulfide bond when the channel is in the closed state (C0), and this locks the channel closed.
X
ABCC7 p.Lys892Cys 25024266:367:43
status: NEW368 We further tested the spontaneous disulfide bond in D110C/K892C-CFTR with the macropatch technique.
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ABCC7 p.Lys892Cys 25024266:368:58
status: NEW369 We pulled inside-out macropatches from oocytes expressing WT- or D110C/K892C-CFTR and recorded the current in real time during exposure to 1 mM DTT backfilled into the pipette.
X
ABCC7 p.Lys892Cys 25024266:369:71
status: NEW372 For D110C/ K892C-CFTR ATPand PKA-activated channels, but unlike WT-CFTR, D110C/K892C-CFTR currents slowly increased over the full duration of the experiment (&#e07a;20 min; Fig. 11 A, c).
X
ABCC7 p.Lys892Cys 25024266:372:11
status: NEWX
ABCC7 p.Lys892Cys 25024266:372:79
status: NEW373 Both WTand D110C/K892C-CFTR currents could be completely abolished with removal of ATP and PKA from the intracellular solution (Fig. 11 A, d).
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ABCC7 p.Lys892Cys 25024266:373:17
status: NEW374 Representative I-V plots at the outset of recording and at 5 and 20 min in WTand D110C/K892C-CFTR are shown in the middle panel of Fig. 11 A.
X
ABCC7 p.Lys892Cys 25024266:374:87
status: NEW375 Unlike WT-CFTR, D110C/K892C-CFTR exhibited strong inward rectification in symmetrical 150 mM Cl&#e032; solution.
X
ABCC7 p.Lys892Cys 25024266:375:22
status: NEW376 The current increase in D110C/K892C-CFTR after ATP and PKA activation was likely caused by DTT-mediated breaking of disulfide bonds between D110C and K892C, allowing more channels to be activated and resulting in a higher current amplitude.
X
ABCC7 p.Lys892Cys 25024266:376:30
status: NEWX
ABCC7 p.Lys892Cys 25024266:376:150
status: NEW377 Meanwhile, the data also suggest that not all D110C/K892C-CFTR channels formed disulfide bonds in the resting state and that some channels could be activated by ATP and PKA in the absence of DTT.
X
ABCC7 p.Lys892Cys 25024266:377:52
status: NEW378 As a control, in the absence of DTT, D110C/K892C-CFTR macroscopic current reached plateau in &#e07a;5 min (like WT-CFTR) and was maintained or slightly decreased in the next &#e07a;20 min (Fig. S7 A).
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ABCC7 p.Lys892Cys 25024266:378:43
status: NEW384 Figure 11.ߓ D110C forms a spontaneous disulfide bond with K892C when channels are in the closed state.
X
ABCC7 p.Lys892Cys 25024266:384:64
status: NEW385 (A) Representative macropatch currents of WTand D110C/K892C-CFTR were recorded in inside-out mode with symmetrical 150 mM Cl&#e032; solution.
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ABCC7 p.Lys892Cys 25024266:385:54
status: NEW388 I-V plots of currents at times a-c for both WTand D110C/K892C-CFTR are shown in the middle panel.
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ABCC7 p.Lys892Cys 25024266:388:56
status: NEW392 (B) 1 mM DTT further activated D110C/K892C-CFTR current but not WT-CFTR current in TEVC recording condition.
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ABCC7 p.Lys892Cys 25024266:392:37
status: NEW393 Representative traces (left) and summary data (right) for macroscopic currents measured from WTand D110C/K892C-CFTR with addition of 1 mM DTT in the presence of ISO.
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ABCC7 p.Lys892Cys 25024266:393:105
status: NEW397 **, P < 0.01 compared with ISO1 in n = 4 for WT-CFTR and n = 5 for D110C/K892C-CFTR experiments.
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ABCC7 p.Lys892Cys 25024266:397:73
status: NEW398 (C) ISO plus DTT failed to further activate D110C/K892C-CFTR current in oocytes pretreated with DTT in TEVC recording condition.
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ABCC7 p.Lys892Cys 25024266:398:50
status: NEW399 Representative trace (left) and summary data (right) for macroscopic currents measured from D110C/K892C-CFTR with prior addition of 1 mM DTT for 3 min in ND96 solution. Current levels in the summary data are given relative to control conditions before first exposure to ISO and normalized to maximal current in response to ISO1 (Imax).
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ABCC7 p.Lys892Cys 25024266:399:98
status: NEW428 Meanwhile, D110 appears to form a salt bridge with K892 of ECL4 and the D110C/K892C spontaneous disulfide bond can only be formed when the channel is in the C0 state (0-ns snapshot in our molecular dynamics simulation; Rahman et al., 2013).
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ABCC7 p.Lys892Cys 25024266:428:78
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.
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ABCC7 p.Lys892Cys 25024266:430:217
status: NEW467 In the present work, we demonstrated that D110C/K892C-CFTR forms a spontaneous disulfide bond and appeared to lock the channel into the C0 state.
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ABCC7 p.Lys892Cys 25024266:467:48
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.Lys892Cys 25024266:493:180
status: NEW495 The D110C/K892C disulfide bond was maintained regardless of CFTR gating energy until the reducing agent DTT was added to break it.
X
ABCC7 p.Lys892Cys 25024266:495:10
status: NEW497 These data suggest that D110C/K892C-CFTR forms a very strong disulfide bond with dissociation energy that must be greater than that of ATP-dependent NBD dimer formation.
X
ABCC7 p.Lys892Cys 25024266:497:30
status: NEW