ABCMdb

ABCC7 p.Glu116Cys

None has been submitted yet.

Publications

PMID: 25024266 [PubMed] Cui G et al: "Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR."

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). Login to comment
109 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). Login to comment
140 However, under the same conditions, no functional modifications were observed for any of the three ECL1 cysteine mutants studied (D110C-, E116C-, and R117C-CFTR). Login to comment
144 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. Login to comment
170 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. Login to comment
189 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. Login to comment
196 Figure 5.ߓ Effects of 2.5 &#b5;M GlyH-101 on WT-, D110C-, E116C-, and R117C-CFTR. Login to comment
254 alone, R104C/E116C-CFTR exhibited very long stable openings with brief closed states and s1 and s2 subconductance states (Fig. 9 B, control). Login to comment
260 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. Login to comment
269 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. Login to comment
283 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. Login to comment
286 (B) Two cysteines engineered at positions 104 and 116 (R104C/E116C) form a spontaneous disulfide bond when CFTR is in the open state. Login to comment
287 Representative single-channel trace of R104C/E116C-CFTR recorded with the same conditions as A. Login to comment
290 +DTT in pipette: R104C/E116C-CFTR recorded with 1 mM DTT in the extracellular pipette solution (left, middle trace). Login to comment
291 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). Login to comment
293 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. Login to comment
294 (C) Cross-linking R104C to E116C using MTS-2-MTS locks CFTR channels into the closed state. Login to comment
295 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. Login to comment
317 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. Login to comment
318 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). Login to comment
319 The R104C/E116C spontaneous open state disulfide bond exhibited the following characteristics: (a) R104C/ E116C-CFTR still required ATP and PKA for activation. Login to comment
320 (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. Login to comment
323 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. Login to comment
324 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. Login to comment
328 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. Login to comment
422 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. Login to comment
430 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. Login to comment
493 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). Login to comment
496 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. Login to comment
498 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. Login to comment