ABCMdb

PMID: 25024266

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., [PubMed]

Sentences

No. Mutations Sentence Comment

15 ABCC7 p.Arg117His ABCC7 p.Arg117Cys ABCC7 p.Glu116Lys ABCC7 p.Asp110His Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. Login to comment 20 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala 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&#e039;-(&#e062;,&#e067;-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. Login to comment 29 ABCC7 p.Arg117His Sheppard et al. (1993) demonstrated that R117H exhibits reduced single-channel conductance, altered sensitivity to external pH, and altered single-channel kinetics resulting in reduction of macroscopic current. Login to comment 31 ABCC7 p.Arg117His ABCC7 p.Glu116Lys ABCC7 p.Asp110His H&#e4;mmerle et al. (2001) reported that mutations D110H, E116K, and R117H induce no trafficking defect when expressed in baby hamster kidney (BHK) cells but affect channel function significantly. Login to comment 32 ABCC7 p.Arg117His ABCC7 p.Glu116Lys ABCC7 p.Asp110His When studied in planar lipid bilayers, R117H-CFTR had gating kinetics similar to WT-CFTR, but a reduced single-channel conductance, whereas D110H- and E116K- CFTR displayed unstable channel openings, leading the authors to propose that ECL1 might contribute to maintaining the open pore architecture of CFTR (H&#e4;mmerle et al., 2001). Login to comment 36 ABCC7 p.Ser108Phe ABCC7 p.Tyr109Cys ABCC7 p.Pro111Ala ABCC7 p.Glu116Lys ABCC7 p.Asp110His CF-causing mutations have been identified in ECL1, including S108F, Y109C/N, D110H/ Y/N,P111A/L,E116K/Q,andR117C/G/H/P/L.Among these residues, D110, E116, and R117 are charged amino acids fully conserved among nine species (Fig. 1 A). Login to comment 37 ABCC7 p.Arg117His Mutations at each of these sites are associated with milder forms of CF; R117H in particular is associated with pancreatic sufficiency and is a common mutation related to mild CF (Kristidis et al., 1992; Sheppard et al., 1993). Login to comment 74 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala Online supplemental material Fig. S1 illustrates representative single-channel current traces of D110R-, E116R-, and R117A-CFTR with a larger time scale (b) whether and how they contribute to maintaining open pore architecture; and (c) whether ECL1 moves during the CFTR gating cycle. Login to comment 93 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 97 ABCC7 p.Arg104Cys ABCC7 p.Arg117Ala ABCC7 p.Glu116Cys 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 100 ABCC7 p.Arg334Cys AMP-PNP also locks mutant R334C-CFTR into a long stable s2 state (Fuller et al., 2005; Zhang et al., 2005b). Login to comment 101 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala Given the instability of D110R-, E116R-, and R117A-CFTR single-channel openings, we asked whether AMP-PNP would lock these mutants into a stable open state. Login to comment 103 ABCC7 p.Arg117Ala In contrast, AMP-PNP did not affect mean burst duration or single-channel amplitude of R117A-CFTR but did increase the apparent open probability (P < 0.05; Fig. 3). Login to comment 104 ABCC7 p.Asp110Arg ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Glu116Arg Similar results were seen in D110R- and E116R-CFTR (mean burst duration for E116R: &#e032;AMP-PNP, 37.72 &#b1; 3.07 ms; +AMP-PNP, 36.16 &#b1; 5.73 ms, n = 3; and for D110R-CFTR: &#e032;AMP-PNP, 22.24 &#b1; 1.8 ms; +AMP-PNP, 19.74 &#b1; 0.69 ms, n = 4). Login to comment 109 ABCC7 p.Arg117Cys ABCC7 p.Arg334Cys ABCC7 p.Asp110Cys ABCC7 p.Glu116Cys 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 113 ABCC7 p.Arg334Cys In contrast, both ET+ and ES&#e032; induced functional covalent modification of R334C-CFTR: ET+ increased macroscopic current by 39% compared with Fig. 2 A. Login to comment 115 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala Fig. S3 shows representative I-V curves of D110R-, E116R-, and R117A-CFTR recorded in symmetrical 150 mM Cl&#e032; solution with the inside-out macropatch technique. Login to comment 117 ABCC7 p.Arg104Glu ABCC7 p.Lys892Glu ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg Fig. S5 illustrates representative single-channel current traces of E116R/ K892E- and R104E/D110R-CFTR and their mean burst durations. Login to comment 118 ABCC7 p.Arg104Glu ABCC7 p.Arg117Glu ABCC7 p.Glu116Arg ABCC7 p.Glu1126Arg Fig. S6 shows representative I-V plots of double mutants R104E/ E116R- and R117E/E1126R-CFTR and their rectification ratio. Login to comment 119 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys Fig. S7 shows representative TEVC current traces of WTand D110C/ K892C-CFTR with DTT pretreatment. Login to comment 124 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala ABCC7 p.Asp112Arg ABCC7 p.Glu115Arg ABCC7 p.Lys114Asp To probe the potential mechanisms by which mutation of these charged residues leads to CF, we first recorded the single-channel behavior of a series of CFTR channel mutants bearing a single mutation at one of the six charged sites (D110R, D112R, K114D, E115R, E116R, or R117A). Login to comment 128 ABCC7 p.Arg117Glu No current was detected in either inside-out patch or TEVC recording from oocytes expressing R117E-CFTR; therefore, it was not studied further. Login to comment 129 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala Unlike WT-CFTR, which opens mainly to the full open state(f)withsubconductancestatesasrareevents,D110R-, E116R-, and R117A-CFTR exhibited multiple open states, including subconductance state 1 (s1), subconductance state 2 (s2), and the f state (Fig. S1). Login to comment 130 ABCC7 p.Arg347Cys ABCC7 p.Arg334Cys ABCC7 p.Arg352Ala This is similar to our previous findings for TM6 mutants R334C-, R352A-, R347C/H-CFTR (Cotten and Welsh, 1999; Zhang et al., 2005b; Cui et al., 2008). Login to comment 132 ABCC7 p.Asp112Arg ABCC7 p.Glu115Arg In contrast, D112R- and E115R-CFTR each mainly opened to the full open state with subconductance states appearing as rare events and mean burst durations very close to that of WT-CFTR. Login to comment 133 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala ABCC7 p.Lys114Asp The exception was K114D-CFTR, which exhibited mean burst duration significantly shorter than that of WT-CFTR, but much longer than that of D110R-, E116R-, and R117A-CFTR. Login to comment 134 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg The apparent open probabilities of D110R-, E116R-, and in these positions were modified by ET+ and ES&#e032; , but their modification failed to affect ion conduction because these amino acids are located too far away from the Cl&#e032; conduction pathway. Login to comment 140 ABCC7 p.Arg117Cys ABCC7 p.Asp110Cys ABCC7 p.Glu116Cys 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 141 ABCC7 p.Arg117Cys These data differ from those of Zhou et al. (2008) who reported that R117C-CFTR could be modified by both ET+ and ES&#e032; . Login to comment 143 ABCC7 p.Arg117Cys Zhou et al. (2008) preincubated cells expressing R117C-CFTR in solution with ET+ or ES&#e032; before the experiment and compared the data with a group of unexposed cells, whereas we investigated the effects of modification in real time and compared data before and after exposure to the reagents in the same oocytes. Login to comment 144 ABCC7 p.Arg117Cys ABCC7 p.Asp110Cys ABCC7 p.Glu116Cys 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 145 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala ABCC7 p.Asp112Arg ABCC7 p.Glu115Arg ABCC7 p.Lys114Asp (A) Representative single-channel current traces and their all-points histograms for WT-, D110R-, D112R-, K114D-, E115R-, E116R-, and R117A-CFTR from inside-out membrane patches excised from Xenopus oocytes, with symmetrical 150 mM Cl&#e032; solution in the presence of 1 mM MgATP and 50 U/ml PKA. Login to comment 149 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala ABCC7 p.Asp112Arg ABCC7 p.Glu115Arg ABCC7 p.Lys114Asp (B and C) Single-channel amplitudes of the full open state (B) and mean burst durations (C) of WT-, D110R-, D112R-, K114D-, E115R-, E116R-, and R117A-CFTR. Login to comment 150 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala (D) Apparent open probability of WT-, D110R-, E116R-, and R117A-CFTR. Login to comment 154 ABCC7 p.Arg334Ala ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala ABCC7 p.Lys114Asp ECL1 mutations shift the reversal potential in macroscopic currents To further verify that these ECL1 amino acids do not strongly or directly affect ion conduction and permeation, we compared the reversal potentials (Vrev) of D110R-, K114D-, E116R-, and R117A-CFTR with WT-CFTR and with the R334A mutant, which has been shown to have a profound effect on Vrev compared with WT-CFTR, consistent with the role of R334 in providing charge in the outer mouth of the open channel. Login to comment 156 ABCC7 p.Arg334Ala ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala E116R- and R117A-CFTR exhibited significantly right-shifted reversal potentials compared with WT-CFTR, but the effects were not as large as for R334A-CFTR. Login to comment 157 ABCC7 p.Asp110Arg ABCC7 p.Lys114Asp In contrast, neither D110R- nor K114D-CFTR altered Vrev. Login to comment 159 ABCC7 p.Arg334Ala As previously reported, R334A-CFTR exhibited outward rectification but with a significantly reduced rectification ratio: 1.33 &#b1; 0.14 (n = 9, P < 0.01). Login to comment 160 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala ABCC7 p.Lys114Asp Of the ECL1 mutants we examined, the rectification ratios for D110R-, K114D-, and R117A-CFTR were similar to WT-CFTR (Fig. 6), whereas E116R-CFTR showed significantly reduced outward rectification. Login to comment 161 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala We also examined the I-V relationship of D110R-, E116R-, and R117A-CFTR with the inside-out macropatch technique in symmetrical that the charged amino acids in ECL1 might be involved in establishing the appropriate architecture for GlyH-101 binding and function. Login to comment 170 ABCC7 p.Arg117Cys ABCC7 p.Asp110Cys ABCC7 p.Glu116Cys 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 172 ABCC7 p.Arg117Ala Figure 3.ߓ R117A-CFTR failed to be locked into a stable open state by AMP-PNP. Login to comment 173 ABCC7 p.Arg117Ala R117A-CFTR was activated with 1 mM Mg-ATP and PKA and recorded under control conditions with ATP + PKA (&#e032;AMP-PNP), followed by addition of 2.75 mM AMP-PNP (+AMP-PNP). Login to comment 176 ABCC7 p.Arg117Ala AMP-PNP had no effect on mean burst duration or single-channel amplitude but significantly increased apparent open probability of R117A-CFTR (n = 4). Login to comment 182 ABCC7 p.Asp110Glu ABCC7 p.Arg117Lys ABCC7 p.Glu116Asp We generated D110E-, E116D-, and R117K-CFTR and observed their single-channel behavior with the inside-out patch technique in symmetrical 150 mM Cl&#e032; solution at VM = &#e032;100 mV. Login to comment 184 ABCC7 p.Asp110Glu ABCC7 p.Asp110Arg D110E-CFTR exhibited a much more stable full open state with mean burst duration significantly longer than D110R-CFTR (P < 0.001; Fig. 7, A and E). Login to comment 185 ABCC7 p.Glu116Arg ABCC7 p.Glu116Asp Similar results were observed when the burst durations of E116D-CFTR and E116R-CFTR were compared (P < 0.001; Fig. 7, B and E). Login to comment 187 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala Under these conditions, E116R-CFTR exhibited slight inward rectification, but both D110R- and R117A-CFTR exhibited linear I-V relationships like that of WT-CFTR. Login to comment 189 ABCC7 p.Arg117Cys ABCC7 p.Arg334Cys ABCC7 p.Asp110Cys ABCC7 p.Glu116Cys 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 ABCC7 p.Arg117Cys ABCC7 p.Asp110Cys ABCC7 p.Glu116Cys Figure 5.ߓ Effects of 2.5 &#b5;M GlyH-101 on WT-, D110C-, E116C-, and R117C-CFTR. Login to comment 204 ABCC7 p.Arg117Glu ABCC7 p.Arg117Glu ABCC7 p.Arg117Glu ABCC7 p.Asp110Arg ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Glu116Arg To test this hypothesis, we first made the mutants E116R/R117E-, D110R/R117E-, and D110R/E116R/R117E-CFTR and studied their single-channel properties. Login to comment 206 ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala All three mutants exhibited very brief openings to the s1, s2, and f states, with mean burst durations significantly lower than that of WT-CFTR (P < 0.001; Fig. 8 B) and R117A-CFTR (Fig. 2 C), but not different from D110R-CFTR and E116R-CFTR (Fig. 2 C). Login to comment 213 ABCC7 p.Arg117Ala ABCC7 p.Arg117Lys was less marked when R117K- and R117A-CFTR were compared (P < 0.05; Fig. 7, C and E). Login to comment 218 ABCC7 p.Asp110Glu ABCC7 p.Arg117Lys ABCC7 p.Glu116Asp D110, E116, and R117 do not interact with each other locally Because charge-retaining ECL1 amino acid mutations D110E, E116D, and R117K partially rescued a steady Figure 6.ߓ Some ECL1 mutants exhibit altered rectification. Login to comment 219 ABCC7 p.Glu116Arg ABCC7 p.Lys114Asp WT-CFTR, K114D-CFTR, and E116R-CFTR currents were generated under a voltage protocol wherein membrane potential was held at 0 mV for 50 ms then ramped from &#e032;100 mV to 100 mV over 300 ms with the TEVC technique. Login to comment 224 ABCC7 p.Arg334Ala ABCC7 p.Arg104Glu ABCC7 p.Arg104Glu ABCC7 p.Arg117Glu ABCC7 p.Lys892Glu ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala ABCC7 p.Glu1126Arg ABCC7 p.Glu1126Arg ABCC7 p.Lys114Asp ABCC7 p.Asp891Cys ABCC7 p.Arg899Cys ABCC7 p.Glu1124Arg Tab l e 1 Reversal potentials of WT-CFTR and mutants in ND96 bath solution CFTR n Vrev mV WT 14 &#e032;27.75 &#b1; 0.78 R334A 6 &#e032;12.15 &#b1; 1.64a R117A 6 &#e032;22.51 &#b1; 0.85a E116R 5 &#e032;21.45 &#b1; 1.14a K114D 5 &#e032;24.68 &#b1; 3.22 D110R 5 &#e032;27.64 &#b1; 3.29 R104E 5 &#e032;21.15 &#b1; 1.08a R899C 4 &#e032;25.30 &#b1; 3.94 D891C 6 &#e032;25.81 &#b1; 2.44 K892E 5 &#e032;23.70 &#b1; 3.62 E1124R 5 &#e032;18.32 &#b1; 0.43a E1126R 5 &#e032;20.67 &#b1; 3.16b R117E/E1126R 6 &#e032;23.06 &#b1; 1.37b R104E/E116R 6 &#e032;27.17 &#b1; 1.08 Values are mean &#b1; SEM. Login to comment 233 ABCC7 p.Arg104Glu ABCC7 p.Arg104Glu ABCC7 p.Arg104Glu ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg E116 forms a salt bridge with R104 in the open state as well as in the closed state To test the above prediction that R104 is a partner for E116, we studied the single mutant R104E-CFTR and the charge-swap double mutants R104E/E116R- and R104E/D110R-CFTR. Login to comment 235 ABCC7 p.Arg104Glu ABCC7 p.Arg104Glu ABCC7 p.Glu116Arg Both R104E-CFTR and R104E/E116R-CFTR exhibited reduced outward rectification with similar reversal potentials, both significantly different from WT-CFTR (Figs. Login to comment 237 ABCC7 p.Arg104Glu Single-channel recording of R104E-CFTR (representative trace in Fig. 9 A) revealed Instead, we found that mutations at E217, D891, R899, or E1124 had no significant effect on single-channel behavior (Fig. S4 A, bottom). Login to comment 238 ABCC7 p.Asp891Cys ABCC7 p.Arg899Cys ABCC7 p.Glu1124Cys We did not observe any evidence of covalent modification of E1124C-, D891C-, or R899C- CFTR; there was no change in current upon exposure to either MTSET+ or MTSES&#e032; . Login to comment 239 ABCC7 p.Glu1124Cys E1124C-CFTR current was transiently increased by MTSET+ , but in a noncovalent manner (Fig. S4 B). Login to comment 240 ABCC7 p.Asp891Cys ABCC7 p.Glu1124Arg ABCC7 p.Glu1124Arg Mutants D891C- and E1124R- CFTRexhibitedslightchangesinrectificationcompared with WT-CFTR (Fig. S4 C), and reversal potentials were also similar to that of WT-CFTR; the reversal potential of E1124R-CFTR was shifted toward zero (Table 1). Login to comment 242 ABCC7 p.Arg899Cys However, in our hands, the R899C mutation had no effect on channel function. Login to comment 246 ABCC7 p.Asp110Glu ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala ABCC7 p.Arg117Lys ABCC7 p.Glu116Asp (A-C) Representative single-channel currents of D110R- and D110E- (A), E116R- and E116D- (B), and R117A- and R117K-CFTR (C) recorded under the same conditions as Fig. 2 A. Login to comment 248 ABCC7 p.Asp110Glu ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala ABCC7 p.Arg117Lys ABCC7 p.Glu116Asp (D) Mean single-channel amplitude of WT-, D110R-, D110E-, E116R-, E116D-, R117A-, and R117K-CFTR. Login to comment 250 ABCC7 p.Asp110Glu ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala ABCC7 p.Arg117Lys ABCC7 p.Glu116Asp (E) Mean burst duration of WT-, D110R-, D110E-, E116R-, E116D-, R117A-, and R117K-CFTR. Login to comment 251 ABCC7 p.Asp110Glu ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala ABCC7 p.Arg117Lys ABCC7 p.Glu116Asp #, P < 0.001 indicates differences between D110R- and D110E-CFTR or E116R- and E116D-CFTR; *, P < 0.05 indicates differences between R117A- and R117K-CFTR. Login to comment 254 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys 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 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys 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 264 ABCC7 p.Arg104Glu ABCC7 p.Glu116Arg Mean burst duration of R104E-CFTR was 325 &#b1; 54.08 ms, significantly shorter than WT but significantly longer than E116R-CFTR (Fig. 9 A, right). Login to comment 265 ABCC7 p.Arg104Glu ABCC7 p.Arg104Glu ABCC7 p.Glu116Arg ABCC7 p.Glu116Arg Single-channel behavior of the double mutant R104E/ E116R-CFTR was similar to R104E-CFTR, with a long, stable f open state and a mean burst duration significantly longer than that of E116R-CFTR (Fig. 9 A, right). Login to comment 266 ABCC7 p.Glu116Arg Although the charge swap mutant did not fully recover WT-CFTR behavior, its recovery compared with E116R-CFTR suggests that E116 may form a salt bridge with R104 when CFTR channels are in the open state. Login to comment 267 ABCC7 p.Arg104Glu ABCC7 p.Asp110Arg ABCC7 p.Asp110Arg In contrast, single-channel properties of the R104E/D110R-CFTR double mutant were not significantly different from the D110R-CFTR single mutant (Fig. S5), suggesting no interaction between R104 and D110. Login to comment 268 ABCC7 p.Arg352Cys ABCC7 p.Asp993Cys 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). Login to comment 269 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys 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 271 ABCC7 p.Arg117Glu ABCC7 p.Arg117Glu ABCC7 p.Arg117Glu ABCC7 p.Asp110Arg ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Glu116Arg (A) Representative single-channel currents of R117E/ E116R-, R117E/D110R-, and R117E/E116R/ D110R-CFTR and corresponding all-points amplitude histograms recorded under the same conditions as Fig. 2 A. Login to comment 274 ABCC7 p.Arg117Glu ABCC7 p.Arg117Glu ABCC7 p.Arg117Glu ABCC7 p.Asp110Arg ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Glu116Arg (C) Mean single-channel amplitudes of WT-, R117E/ E116R-, R117E/D110R-, and R117E/E116R/ D110R-CFTR. Login to comment 283 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys 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 284 ABCC7 p.Arg104Glu ABCC7 p.Arg104Glu ABCC7 p.Glu116Arg (A) Representative single-channel current traces of R104E- and R104E/E116R-CFTR recorded with the same experimental conditions as Fig. 2 (left), their all-points amplitude histograms (middle), and mean burst durations (right). Login to comment 285 ABCC7 p.Arg104Glu ABCC7 p.Glu116Arg ABCC7 p.Glu116Arg **, P < 0.01 indicates a significant difference between E116R- and R104E/ E116R-CFTR. Login to comment 286 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys (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 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys Representative single-channel trace of R104C/E116C-CFTR recorded with the same conditions as A. Login to comment 290 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys +DTT in pipette: R104C/E116C-CFTR recorded with 1 mM DTT in the extracellular pipette solution (left, middle trace). Login to comment 291 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys 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 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys 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 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys (C) Cross-linking R104C to E116C using MTS-2-MTS locks CFTR channels into the closed state. Login to comment 295 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys 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 301 ABCC7 p.Lys892Glu ABCC7 p.Lys892Glu ABCC7 p.Asp110Arg To test this prediction, we made the single mutation K892E and the double mutation D110R/K892E. Login to comment 302 ABCC7 p.Lys892Glu K892E-CFTR open channels behaved similarly to WT-CFTR, including displaying a stable full open state with single-channel amplitude similar to WT (&#e032;0.77 &#b1; 0.02 pA, n = 5; Fig. 10), outward rectification in the I-V relationship, and WT-like reversal potential (Fig. S4 C and Table 1). Login to comment 304 ABCC7 p.Lys892Glu ABCC7 p.Asp110Arg ABCC7 p.Asp110Arg The double mutant D110R/K892E-CFTR behaved similarly to D110R-CFTR (Fig. 7 A), displaying flickery openings to the s1, s2, and f states with a very brief open burst duration (Fig. 10 A). Login to comment 310 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys To test this, we made the double cysteine mutant D110C/K892C-CFTR. Login to comment 311 ABCC7 p.Asp110Arg Its single-channel behavior was very similar to that of D110R-CFTR except with a longer mean burst duration, probably because the cysteine at position 110 carries a partial negative charge that stabilizes channel open pore architecture in the same manner as D110 does (Fig. 10 A). Login to comment 312 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 313 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 317 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys 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 ABCC7 p.Glu116Cys 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 ABCC7 p.Arg104Cys ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys ABCC7 p.Glu116Cys 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 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys (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 321 ABCC7 p.Arg352Cys ABCC7 p.Arg334Cys ABCC7 p.Asp993Cys ABCC7 p.Glu217Cys 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). Login to comment 323 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys 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 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys 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 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys 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 329 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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). Login to comment 332 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 335 ABCC7 p.Lys892Glu ABCC7 p.Lys892Glu ABCC7 p.Asp110Cys ABCC7 p.Asp110Arg ABCC7 p.Lys892Cys (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). Login to comment 336 ABCC7 p.Lys892Glu ABCC7 p.Asp110Cys ABCC7 p.Asp110Arg ABCC7 p.Asp110Arg ABCC7 p.Asp110Arg ABCC7 p.Lys892Cys *, P < 0.05 indicates a significant difference between D110R- and D110R/K892E-CFTR; #, P < 0.001 for D110C/K892C-CFTR compared with D110R alone. Login to comment 342 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys (C) Exposure to 1 mM DTT, backfilled into the pipette, increased the number of active channels in D110C/K892C-CFTR. Login to comment 347 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 350 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys In contrast, in D110C/K892C-CFTR, activation by ISO plus DTT (ISO2 + DTT) led to significantly higher current than ISO alone (ISO1). Login to comment 351 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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). Login to comment 353 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys We then asked whether DTT could further activate D110C/K892C-CFTR current if we used DTT before ISO. Login to comment 354 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys We used 1 mM DTT alone for 3 min followed by ISO alone (ISO1), which fully activated D110C/K892C-CFTR channels to plateau. Login to comment 357 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 361 ABCC7 p.Asp110Cys ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys ABCC7 p.Lys892Cys 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. Login to comment 362 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 363 ABCC7 p.Asp110Cys ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys ABCC7 p.Lys892Cys 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. Login to comment 364 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 367 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 368 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys We further tested the spontaneous disulfide bond in D110C/K892C-CFTR with the macropatch technique. Login to comment 369 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 372 ABCC7 p.Asp110Cys ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys ABCC7 p.Lys892Cys 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). Login to comment 373 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys Both WTand D110C/K892C-CFTR currents could be completely abolished with removal of ATP and PKA from the intracellular solution (Fig. 11 A, d). Login to comment 374 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 375 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys Unlike WT-CFTR, D110C/K892C-CFTR exhibited strong inward rectification in symmetrical 150 mM Cl&#e032; solution. Login to comment 376 ABCC7 p.Asp110Cys ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys ABCC7 p.Lys892Cys 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. Login to comment 377 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 378 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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). Login to comment 380 ABCC7 p.Arg117Glu ABCC7 p.Glu1126Arg ABCC7 p.Glu1126Arg Sample current traces for both E1126R- and R117E/ E1126R-CFTR are shown in Fig. 12 A. Login to comment 381 ABCC7 p.Arg117Glu As mentioned previously, we did not detect current from R117E-CFTR We also tested the possibility that E116 could form an open state salt bridge with K892. Login to comment 382 ABCC7 p.Lys892Glu ABCC7 p.Glu116Arg E116R/K892E-CFTR exhibited an I-V relationship similar to that of WT-CFTR. Login to comment 383 ABCC7 p.Lys892Glu ABCC7 p.Glu116Arg ABCC7 p.Glu116Arg The burst behavior of E116R/K892E-CFTR was similar to that of E116R-CFTR, suggesting that E116 does not form a salt bridge with K892 when the CFTR channel is in the open state (Fig. S5). Login to comment 384 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys Figure 11.ߓ D110C forms a spontaneous disulfide bond with K892C when channels are in the closed state. Login to comment 385 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys (A) Representative macropatch currents of WTand D110C/K892C-CFTR were recorded in inside-out mode with symmetrical 150 mM Cl&#e032; solution. Login to comment 388 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys I-V plots of currents at times a-c for both WTand D110C/K892C-CFTR are shown in the middle panel. Login to comment 392 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys (B) 1 mM DTT further activated D110C/K892C-CFTR current but not WT-CFTR current in TEVC recording condition. Login to comment 393 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 397 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys **, P < 0.01 compared with ISO1 in n = 4 for WT-CFTR and n = 5 for D110C/K892C-CFTR experiments. Login to comment 398 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys (C) ISO plus DTT failed to further activate D110C/K892C-CFTR current in oocytes pretreated with DTT in TEVC recording condition. Login to comment 399 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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). Login to comment 402 ABCC7 p.Arg117Cys ABCC7 p.Arg117Cys ABCC7 p.Glu1126Cys The single-channel amplitudes of R117C- and R117C/E1126C-CFTR were slightly, but significantly, smaller than that of WT-CFTR (Fig. 12 C). Login to comment 403 ABCC7 p.Arg117Glu ABCC7 p.Arg117Glu ABCC7 p.Glu1126Arg ABCC7 p.Glu1126Arg ABCC7 p.Glu1126Arg ABCC7 p.Glu1126Arg We also compared the fractional burst duration represented by s1, s2, and f states for E1126R- and R117E/E1126R-CFTR and found that E1126R exhibited a significantly higher fraction of both s1 and s2 states than WT, whereas the distribution for double mutant R117E/E1126R-CFTR was similar to WT-CFTR, exhibiting mainly the f state (a nearly complete rescue; Fig. 12 D). Login to comment 405 ABCC7 p.Glu1126Arg E1126R-CFTR mainly opened to the full open state but with frequent brief transitions to the s1 and s2 states. Login to comment 406 ABCC7 p.Arg117Glu ABCC7 p.Arg117Ala ABCC7 p.Glu1126Arg R117E/E1126R-CFTR opened to a full open state much more often compared with R117A-CFTR (Fig. 2). Login to comment 407 ABCC7 p.Arg117Glu ABCC7 p.Glu1126Arg ABCC7 p.Glu1126Arg Mean burst duration for both E1126R- and R117E/ E1126R-CFTR are summarized in Fig. 12 B. Login to comment 408 ABCC7 p.Arg117Cys ABCC7 p.Arg117Glu ABCC7 p.Arg117Ala ABCC7 p.Glu1126Arg The mean burst duration of R117E/E1126R-CFTR was significantly longer than that of R117A- and R117C-CFTR. Login to comment 409 ABCC7 p.Arg104Glu ABCC7 p.Glu116Arg However, as noted previously for R104E/E116R-CFTR, the charge swap mutant did not completely recover the behavior of Figure 12.ߓ R117 forms a salt bridge with E1126 in the open state. Login to comment 410 ABCC7 p.Arg117Cys ABCC7 p.Arg117Cys ABCC7 p.Arg117Glu ABCC7 p.Glu1126Arg ABCC7 p.Glu1126Arg ABCC7 p.Glu1126Cys (A) Representative single-channel current traces of E1126R-, R117E/E1126R-, R117C-, and R117C/E1126C-CFTR recorded under the same experimental conditions as Fig. 2 and their all-points amplitude histograms (right). Login to comment 412 ABCC7 p.Arg117Cys ABCC7 p.Arg117Cys ABCC7 p.Arg117Cys ABCC7 p.Arg117Cys ABCC7 p.Arg117Glu ABCC7 p.Glu1126Arg ABCC7 p.Glu1126Arg ABCC7 p.Glu1126Cys #, P < 0.01 indicates a significant difference between WTand R117C-CFTR; **, P < 0.01 indicates a significant difference between WTand E1126R-CFTR, between R117C and R117E/E1126R-CFTR, and between R117C and R117C/E1126C. Login to comment 413 ABCC7 p.Arg117Glu n.d., no current detected for R117E-CFTR in Xenopus oocytes. Login to comment 417 ABCC7 p.Arg117Glu ABCC7 p.Glu1126Arg ABCC7 p.Glu1126Arg (D) Mean fraction of open burst duration is plotted for each open conductance state of E1126R- and R117E/E1126R-CFTR. Login to comment 422 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys 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 423 ABCC7 p.Asp110Glu ABCC7 p.Asp110Arg ABCC7 p.Glu116Arg ABCC7 p.Arg117Ala ABCC7 p.Arg117Lys ABCC7 p.Glu116Asp As shown here, mean burst durations of charge-retaining mutants D110E-, E116D-, and R117K-CFTR are significantly longer than their related charge-reversing or charge-destroying mutants D110R-, E116R-, and R117A-CFTR but distinctly shorter than that of WT-CFTR (Fig. 7). Login to comment 428 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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). Login to comment 430 ABCC7 p.Arg104Cys ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys ABCC7 p.Glu116Cys 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 431 ABCC7 p.Arg117Cys ABCC7 p.Arg117Cys ABCC7 p.Glu1126Cys R117C/E1126C-CFTR exhibited very brief openings to multiple open states, including s1, s2, and f, with significantly shorter mean burst duration compared with R117C-CFTR (P < 0.01), likely caused by mutual repulsion by the partial negative charges at the two cysteines, leading to unstable open states (Fig. 12, A and B). Login to comment 437 ABCC7 p.Lys892Glu Top view of the homology model (McCarty laboratory model; Rahman et al., 2013) with salt bridge residues shown as spheres: R117-E1126, red; E116-R104, green; and D110-K892E, magenta. Login to comment 467 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 493 ABCC7 p.Arg104Cys ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys ABCC7 p.Glu116Cys 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 495 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys The D110C/K892C disulfide bond was maintained regardless of CFTR gating energy until the reducing agent DTT was added to break it. Login to comment 496 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys 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 497 ABCC7 p.Asp110Cys ABCC7 p.Lys892Cys 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. Login to comment 498 ABCC7 p.Arg104Cys ABCC7 p.Glu116Cys 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