ABCMdb

PMID: 16227620

Zhang ZR, Song B, McCarty NA
State-dependent chemical reactivity of an engineered cysteine reveals conformational changes in the outer vestibule of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2005 Dec 23;280(51):41997-2003. Epub 2005 Oct 14., 2005-12-23 [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Arg334Cys In single R334C-CFTR channels studied in excised patches, modification by [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET؉ ), which increases conductance, occurred only during channel closed states. Login to comment 4 ABCC7 p.Arg334Cys R334C-CFTR channels in outside-out macropatches activated by ATP alone were modified with first order kinetics upon rapid exposure to MTSET؉ . Login to comment 5 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys Modification was much slower when channels were locked open by the addition of nonhydrolyzable nucleotide or when the R334C mutation was coupled to a second mutation, K1250A, which greatly decreases channel closing rate. Login to comment 6 ABCC7 p.Lys464Ala ABCC7 p.Arg334Cys In contrast, modification was faster in R334C/K464A-CFTR channels, which exhibit prolonged interburst closed states. Login to comment 7 ABCC7 p.Arg334Cys These data indicate that the reactivity of the engineered cysteine in R334C-CFTR is state-dependent, providing evidence of changes in pore conformation coupled to ATP binding and hydrolysis at the NBDs. Login to comment 8 ABCC7 p.Arg334Cys The data also show that maneuvers that lock open R334C-CFTR do so by locking channels into the prominent s2 subconductance state, suggesting that the most stable conducting state of the pore reflects the fully occupied, prehydrolytic state of the NBDs. Login to comment 16 ABCC7 p.Arg334Cys In that study, real time modification of single R334C-CFTR channels was observed during patch clamp experiments by the sulfhydryl-modifying agent, MTSETϩ , diffusing to the tip of the electrode; the resulting deposition of positive charge increased the open channel conductance. Login to comment 18 ABCC7 p.Arg334Cys Therefore, we hypothesized that the accessibility or reactivity of the engineered cysteine in R334C-CFTR for modification by MTSETϩ may be favored by the closed state. Login to comment 19 ABCC7 p.Arg334Cys To test this hypothesis, we performed a series of experiments to measure the rate coefficients for modification by MTSETϩ and MTSES- under a variety of conditions that alter the channel open probability (Po) of R334C-CFTR. Login to comment 22 ABCC7 p.Arg334Cys EXPERIMENTAL PROCEDURES Preparation of Oocytes and cRNA-For mutant R334C, site-directed mutagenesis used a nested PCR strategy in which the mutation was designed into antiparallel oligomers (18). Login to comment 40 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys All single channel recordings for R334C- and R34C/K1250A-CFTR used asymmetrical [Cl- ] in order to increase the single channel amplitude at VM ϭ -100 mV, where the pipettes were filled with a low [Cl- ]-containing solution (see below). Login to comment 45 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys R334C-, R334C/ K464A-, and R334C/K1250A-CFTR channels were activated by excision into intracellular solution containing 300 mM NMDG-Cl, 1.1 mM MgCl2, 2 mM Tris-EGTA, 1 mM MgATP, 10 mM TES (pH 7.4), 50 units/ml PKA. Login to comment 46 ABCC7 p.Arg334Cys In experiments designed to increase Po of R334C-CFTR channels, 2.75 mM AMP-PNP was added to intracellular solution containing 1 mM MgATP and 100 units/ml PKA. Login to comment 60 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys The bin widths for R334C-CFTR recordings in the absence of AMP-PNP were 100 ms, and for R334C-CFTR in the presence of additional AMP-PNP or for a dual mutant R334C/K1250A, bin widths were 500 and 1000 ms, respectively. Login to comment 71 ABCC7 p.Arg334Cys RESULTS R334C Channels Are Modified by MTSETϩ Only in the Closed State-We have shown previously that the MTSETϩ -induced covalent modification of a cysteine engineered at CFTR position 334 (in transmembrane domain 6) increased single channel conductance without altering gating properties (17, 18). Login to comment 75 ABCC7 p.Arg334Cys Despite hours of recording of R334C-CFTR channels, the process of modification was observed to occur only during the closed interval (i.e. sometime between the last opening with lower conductance and the first opening with higher conductance). Login to comment 76 ABCC7 p.Arg334Cys This observation led us to hypothesize that modification of R334C-CFTR might be favored by the closed state. Login to comment 77 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys To test this hypothesis, we coupled the R334C mutation with a mutation at the Walker lysine of NBD2, K1250A, which prolongs the open burst duration of CFTR channels (21-23). Login to comment 78 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys Fig. 1B shows a recording of a single R334C/ K1250A-CFTR channel, where the electrode was backfilled with 200 ␮M MTSETϩ . Login to comment 81 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys Hence, even when Po was increased by the K1250A mutation, modification at R334C did not take place in the open state. Login to comment 82 ABCC7 p.Arg334Cys These observations strongly suggest that modification of R334C-CFTR by MTSETϩ is favored by the closed state. Login to comment 83 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Movement in the Outer Vestibule of the CFTR Pore 41998 As shown in Fig. A and reported previously (17), R334C-CFTR channels exhibit stable subconductance behavior, including transitions to s1, s2, and f conductance states. Login to comment 84 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys In contrast, all of the open bursts of R334C/K1250A-CFTR before modification lacked transitions between conductance states and were "locked" in the s2 state (Fig. 1B). Login to comment 85 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys Following MTSETϩ -induced modification, R334C/K1250A-CFTR channels opened to, and remained locked in, the s2 state. Login to comment 86 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Amplitudes for the s2 state in R334C/K1250A-CFTR were not significantly different from the amplitudes of the s2 state of R334C-CFTR before and after modification (p ϭ 0.85) (17). Login to comment 87 ABCC7 p.Arg334Cys These data suggest that interruption of the ATP-dependent gating cycle leads to stabilization of the pore conformation, resulting in fewer transitions between the three open conductance levels characteristic of R334C-CFTR; this provides further support for the notion that transitions between open conductance levels in CFTR channels are linked to NBD-mediated gating events. Login to comment 88 ABCC7 p.Arg334Cys Macroscopic Kinetics of Modification Were Altered in the Presence of AMP-PNP-If modification of R334C-CFTR is favored by the closed state, we would expect to observe a slowing of the macroscopic time course of modification under conditions that increase Po. Login to comment 89 ABCC7 p.Arg334Cys To test this hypothesis, we studied outside-out macropatches pulled from oocytes expressing R334C-CFTR (17). Login to comment 93 ABCC7 p.Arg334Cys To increase the Po of R334C-CFTR channels, we included a poorly hydrolyzable ATP analogue, AMP-PNP, at 2.75 mM in addition to ATP in the pipette (24, 25). Login to comment 94 ABCC7 p.Arg334Cys Fig. 2B shows that the increase in macroscopic current upon exposure of R334C-CFTR channels to MTSETϩ in the presence of cytosolic ATP ϩ AMP-PNP exhibited a somewhat different time course compared with experiments with ATP alone. Login to comment 97 ABCC7 p.Arg334Cys Hence, modification of R334C-CFTR in the presence of mixtures of ATP and AMP-PNP occurred in two phases. Login to comment 102 ABCC7 p.Arg334Cys To understand better the results from macropatch experiments, we performed detached inside-out single channel recordings in R334C-CFTR, in the presence of ATP ϩ AMP-PNP, using the real time modification approach. Login to comment 107 ABCC7 p.Lys1250Ala Furthermore, the prolonged unmodified FIGURE1.RealtimemodificationofR334C-CFTRandR334C/K1250A-CFTRchannels by MTSET؉ . Login to comment 108 ABCC7 p.Arg334Cys A, a representative trace for R334C-CFTR in the excised, inside-out patch configuration during real time modification by MTSETϩ backfilled in the pipette. Login to comment 111 ABCC7 p.Arg334Cys Current levels for unmodified R334C-CFTR are indicated by the four dashed lines (in order from top to bottom) c, s1, s2, and f, representing the closed, subconductance 1, subconductance 2, and full conductance states. Login to comment 112 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys B, representative trace for R334C/K1250A-CFTR under identical conditions. Login to comment 115 ABCC7 p.Arg334Cys Modification of R334C-CFTR is slowed in the presence of AMP-PNP. Login to comment 116 ABCC7 p.Arg334Cys A, a representative recording of macroscopic current of R334C-CFTR. Login to comment 120 ABCC7 p.Arg334Cys B, a representative recording of macroscopic current from R334C-CFTR in the presence of ATP ϩ AMP-PNP. Login to comment 122 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Movement in the Outer Vestibule of the CFTR Pore DECEMBER 23, 2005•VOLUME 280•NUMBER 51 JOURNAL OF BIOLOGICAL CHEMISTRY 41999 openings and prolonged modified openings induced by AMP-PNP were "locked" in the s state, as was found for R334C/K1250A-CFTR with ATP alone, and the s2 state amplitudes were virtually identical to those for the s2 state of R334C-CFTR in the presence of ATP alone (p Ͼ 0.5). Login to comment 124 ABCC7 p.Arg334Cys We analyzed the mean burst duration of unmodified R334C-CFTR channels either in the presence of ATP alone (Fig. 4A) or in the presence of ATP ϩ AMP-PNP (Fig. 4C). Login to comment 127 ABCC7 p.Arg334Cys In contrast, the histogram for R334C-CFTR channels in the presence of ATP ϩ AMP-PNP was fit best with the sum of two exponential functions having ␶B1 ϭ 0.52 Ϯ 0.16 s (p Ͼ 0.1 compared with ␶B obtained with ATP alone) and ␶B2 ϭ 10.3 Ϯ 1.3 s (Fig. 4D). Login to comment 128 ABCC7 p.Arg334Cys The fractional amplitudes contributing to the fits corresponding to ␶B1 and ␶B2 were 71 and 29%, respectively, which are very similar to the fractional amplitudes for ␶1 and ␶2 obtained from the kinetic analysis of modification of R334C-CFTR macroscopic current by MTSETϩ in the presence of ATP ϩ AMP-PNP (69 Ϯ 4 and 31 Ϯ 4%, respectively). Login to comment 129 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys These data suggest that the faster phase of macroscopic modification arises from modification of R334C-CFTR channels with burst duration of ϳ0.5 s, and the slower phase of macroscopic modification of R334C-CFTR channels reflects the modification of those channels with burst duration of ϳ10 s. Hence, these resultssuggestthatthemodificationratecoefficientslowsunderconditions thatincreasePo,whichisconsistentwiththenotionthatMTSETϩ -induced modification in R334C-CFTR is favored by the closed state. Login to comment 130 ABCC7 p.Lys1250Ala TABLEONE KineticsofmodificationofR334Cunderavarietyofconditions ValuesshownaremeanϮS.E. Condition1mMATP0.2mMATP1mMATP 1mMATP؉2.75mM AMP-PNP 1mMATP1mMATP1mMATP1mMATP MutantR334CR334CR334C/K464AR334CR334C/K1250AR334CR334CR334C/K1250A ͓MTSETϩ ͔(␮M)101010101050 ͓MTSES- ͔(␮M)5050 ␶(s)(n)5.93Ϯ1.37(4)5.88Ϯ0.49(5)2.44Ϯ0.27(3)a 4.35Ϯ0.9and 157Ϯ14.9(5) 12.5Ϯ0.94and 225Ϯ29(5)a 2.73Ϯ0.24(5)4.23Ϯ0.18(3)b 11.3Ϯ0.7and 132Ϯ30.1(5) a SignificantdifferencefromvalueforR334C-CFTRwith1mMATP,exposedto10␮MMTSETϩ . Login to comment 134 ABCC7 p.Arg334Cys This individual patch contained at least three R334C-CFTR channels, recorded in the presence of ATP plus AMP-PNP. Login to comment 144 ABCC7 p.Lys1250Ala Mutation K1250A reduces the channel closing rate (Fig. 1B). Login to comment 145 ABCC7 p.Lys464Ala Mutation K464A in NBD1 leads to a great reduction in the channel opening rate. Login to comment 146 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys We studied outside-out macropatches from oocytes expressing R334C/K1250A- or R334C/K464A-CFTR to determine the effects of these gating domain mutations on the kinetics of modification, using experimental procedures similar to those described above. Login to comment 147 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys Upon exposure to MTSETϩ , the macroscopic current for R334C/K1250A-CFTR increased rapidly at first, followed by a slower increase in current, reflecting a complicated modification process (Fig. 5A); the kinetics of modification were described best by the sum of two exponential functions. Login to comment 148 ABCC7 p.Lys1250Ala Hence, the consequences of introducing the K1250A mutation were similar to the consequences of the addition of nonhydrolyzable nucleotide; the time-course of macroscopic modification was biphasic, with a component that is much slower than that seen in the single mutant with ATP alone. Login to comment 149 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys In five experiments (TABLE ONE), ␶1 averaged 12.5 Ϯ 0.94 s (fractional amplitude 74.7 Ϯ 1.3%), which was significantly larger than the value of ␶ for R334C-CFTR in the presence of ATP alone and the value of ␶1 for R334C-CFTR in the presence of ATP ϩ AMP-PNP (p Ͻ 0.001). Login to comment 150 ABCC7 p.Arg334Cys For those five experiments, ␶2 averaged 225 Ϯ 29 s (fractional amplitude 25.3 Ϯ 1.3%), which was somewhat larger than the value of ␶2 for R334C-CFTR in the presence of ATP ϩ AMP-PNP (p ϭ 0.049). Login to comment 151 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys The modification rate coefficients for MTSETϩ in R334C/K1250A-CFTR were 9,840 Ϯ 626 M -1 s-1 and 482 Ϯ 65 M -1 s-1 , respectively. Login to comment 152 ABCC7 p.Lys1250Ala The biphasic nature of the macroscopic kinetics of modification in these experiments probably reflects the fact that the K1250A mutation reduces the closing rate in some channels but not all (23). Login to comment 153 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys In other words, while R334C/K1250A-CFTR channels are closed, they stay closed approximately as long as R334C-CFTR channels do, which provides an opportunity for rapid modification. Login to comment 154 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys When R334C/K1250A-CFTR channels are open, they typically stay open much longer than R334C-CFTR channels do, which reduces the macroscopic modification rate coefficient. Login to comment 155 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys These interpretations are supported, at least in part, by the single channel behavior of R334C/K1250A-CFTR. Login to comment 157 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys The top, middle, and bottom traces are from near the beginning, middle, and end of the experiment, respectively. One can see that R334C/K1250A-CFTR channel openings lack prominent transitions between conductance states, no matter how long the burst duration. Login to comment 159 ABCC7 p.Arg334Cys In a manner similar to the experiments using R334C-CFTR in the presence of ATP ϩ AMP-PNP, the briefer open bursts were always modified earlier than the longer bursts. Login to comment 161 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys However, we were able to estimate mean burst duration of R334C/K1250A-CFTR channels. Login to comment 163 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys The fractional amplitudes contributed by ␶B1 and ␶B2 were 77 and 23%, respectively, which are very compatible with the fractional amplitudes for ␶1 (75%) and ␶2 (25%) for the kinetics of macroscopic modification of R334C/K1250A-CFTR. Login to comment 164 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys This suggests that the faster phase of macroscopic modification of R334C/K1250A-CFTR by MTSETϩ could be attributed to modification of channels with burst duration of ϳ4 s, FIGURE 5. Login to comment 165 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys MTSET؉ -induced modification of R334C/K1250A-CFTR and R334C/ K464A-CFTR. Login to comment 166 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Shown are outside-out macropatches from oocytes expressing either R334C/K1250A-CFTR (A and C) or R334C/K464A-CFTR (B). Login to comment 169 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys The process of modification of R334C/K1250A-CFTR by MTSETϩ at Vm ϭ ϩ80 mVwasfitbestwiththesumoftwoexponentialfunctions,with␶1 ϭ14.8sand␶2 ϭ158s in this experiment. Login to comment 170 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys The kinetics of modification of R334C/K1250A-CFTR by MTSETϩ at Vm ϭ -80 mV also were described best by the sum of two exponential functions, having values of ␶1 ϭ 12.9 s and ␶2 ϭ 126 s in this experiment. Login to comment 171 ABCC7 p.Lys464Ala ABCC7 p.Arg334Cys In contrast, the kinetics of modification of R334C/K464A-CFTR by MTSETϩ were fit best with a first-order exponential function, with ␶ ϭ 1.92 s in this experiment. Login to comment 173 ABCC7 p.Arg334Cys Effects of AMP-PNP on mean burst duration of unmodified R334C-CFTR channels. Login to comment 174 ABCC7 p.Arg334Cys A, R334C-CFTR channels recorded with ATP and PKA. Login to comment 177 ABCC7 p.Arg334Cys C, after R334C-CFTR channels were activated by ATP and PKA, 2.75 mM AMP-PNP was added to the solution. Login to comment 183 ABCC7 p.Lys1250Ala This behavior also is characteristic of the K1250A single mutant, which under identical conditions exhibits PO of only ϳ0.77 (27). Login to comment 186 ABCC7 p.Arg334Cys These results are consistent with our hypothesis that modification of R334C is favored by the closed state. Login to comment 187 ABCC7 p.Arg334Cys We also reasoned that if modification of R334C-CFTR channels is favored by the closed state, the modification rate coefficient should be higher under conditions that reduce Po. Login to comment 188 ABCC7 p.Arg334Cys To test this hypothesis, we first studied outside-out macropatches of R334C-CFTR channels in the presence of 0.2 mM ATP and measured the kinetics of modification. Login to comment 189 ABCC7 p.Arg334Cys Surprisingly, the time constant of modification was identical to that observed for R334C-CFTR in the presence of 1 mM ATP (p ϭ 0.72; TABLE ONE). Login to comment 190 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys We speculated that 0.2 mM ATP may slightly reduce the Po of R334C-CFTR channels, but not to a degree that could alter significantly the kinetics of modification; indeed, the overall Po of R334C-CFTR channels recorded in the presence of 1 mM ATP is already reduced, compared with WT-CFTR under identical conditions (0.24 Ϯ 0.04 (17) versus 0.34 Ϯ 0.03 (27), respectively). Login to comment 191 ABCC7 p.Lys464Ala ABCC7 p.Arg334Cys Therefore, we recorded from giant outside-out patches pulled from oocytes expressing R334C/ K464A-CFTR, which would reduce Po considerably by prolonging the interburst closed durations (21-23) (Fig. 5B). Login to comment 192 ABCC7 p.Lys464Ala ABCC7 p.Arg334Cys The macroscopic current of R334C/K464A-CFTR was increased rapidly upon application of 10 ␮M MTSETϩ . Login to comment 193 ABCC7 p.Arg334Cys The kinetics of modification were described best by a first-order exponential (TABLE ONE; p ϭ 0.004 compared with ␶ for R334C-CFTR). Login to comment 194 ABCC7 p.Lys464Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys The modification rate coefficient for MTSETϩ in R334C/K464A-CFTR was 41,864 Ϯ 4,229 M -1 s-1 , which is roughly 2-fold higher than that in R334C-CFTR under identical conditions (p ϭ 0.007). Login to comment 196 ABCC7 p.Arg334Cys Kinetics of Modification by MTSES- -Because our previous studies (17, 18) showed that the electrostatic potential in the outer vestibule affects the kinetics of modification at R334C, we asked whether the modification rate coefficient (and its potential state dependence) for a negatively charged thiol-modifying reagent was different from that measured for the positively charged MTSETϩ . Login to comment 197 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Fig. 7 shows outside-out macropatch recordings from oocytes expressing either R334C-CFTR or R334C/K1250A-CFTR, with rapid exposure to 50 ␮M MTSES- . Login to comment 198 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Macroscopic currents from R334C- and R334C/K1250A-CFTR were decreased upon exposure to MTSES- (due to deposition of negative charge) by 75 Ϯ 6 and 77 Ϯ 5%, respectively. Login to comment 199 ABCC7 p.Arg334Cys The kinetics of modification of R334C-CFTR by MTSES- were fit best with a first-order exponential function (TABLE ONE). Login to comment 200 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys The macroscopic kinetics of modification of R334C/K1250A-CFTR were fit best with the sum of two exponential functions (TABLE ONE; the fractional amplitudes were 84 Ϯ 2.7% for ␶1 and 16 Ϯ 2.7% for ␶2), as was found for MTSETϩ . Login to comment 201 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys The modification rate coefficients for MTSES- in both R334C-CFTR and R334C/K1250A-CFTR were Ͼ3-fold lower than those measured for MTSETϩ in the same mutants (p Ͻ 0.001). Login to comment 203 ABCC7 p.Arg334Cys More importantly, the kinetics of modification of the engineered cysteine at R334C by MTSES- were state-dependent, as described above for modification by MTSETϩ . Login to comment 206 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys Upon rapid exposure to MTSETϩ , macroscopic inward current was increased, reflecting modification of R334C/ K1250A-CFTR channels. Login to comment 208 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys Kinetics of modification of single R334C/K1250A-CFTR channels. Login to comment 217 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys All openings of R334C/K1250A-CFTR channels exhibited conductance equivalent to the s2 conductance state of R334C-CFTR. Login to comment 220 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys MTSES- -induced modification of R334C-CFTR and R334C/K1250A-CFTR. Login to comment 221 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Outside-out macropatches were pulled from oocytes expressing either R334C-CFTR or R334C/K1250A-CFTR. Login to comment 223 ABCC7 p.Arg334Cys A, R334C-CFTR channels were activated by ATP plus PKA. Login to comment 225 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys The dashed line indicates a fit of the data to a first-order exponentialfunction,having␶ϭ4.5sinthisexperiment.B,arepresentativerecordingof macroscopic current of R334C/K1250A-CFTR under identical conditions. Login to comment 229 ABCC7 p.Arg334Cys These results indicate that the direction of anion movement does not affect the rate of modification by MTS reagent at R334C. Login to comment 231 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys Single R334C-CFTR channels studied using real time modification only showed a reaction to MTSETϩ during a closed state, even when channel Po was increased dramatically by exposure to mixtures of ATP and AMP-PNP or by the addition of the Walker A mutation K1250A. Login to comment 232 ABCC7 p.Arg334Cys Macropatch currents recorded from oocytes expressing R334C-CFTR increased rapidly upon abrupt exposure to MTSETϩ (or decreased rapidly upon abrupt exposure to MTSES- ). Login to comment 233 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Under conditions that increase channel activity (i.e. R334C-CFTR with ATP ϩ AMP-PNP, or R334C/K1250A-CFTR with ATP alone), the kinetics of modification were slowed. Login to comment 234 ABCC7 p.Lys464Ala ABCC7 p.Arg334Cys Under conditions that decrease channel activity (R334C/K464A-CFTR), the rate of modification was increased dramatically. Login to comment 237 ABCC7 p.Arg334Cys Our data indicate that the rate of covalent modification at R334C differs dramatically between closed and open channels. Login to comment 239 ABCC7 p.Arg334Cys However, we previously found that the macroscopic conductance of whole oocytes expressing R334C-CFTR channels was sensitive to bath pH, due to titration of the partial negative charge on the unmodified cysteine (17, 18). Login to comment 240 ABCC7 p.Arg334Cys This observation suggests that R334C indeed faces the water-soluble pore while the channels are open, because protons can access this residue. Login to comment 241 ABCC7 p.Arg334Cys Hence, the state-dependent ability of MTS reagents to interact with the engineered cysteine of R334C-CFTR most likely reflects a difference in the reactivity of that cysteine during channel closure rather than physical obstruction that reduces accessibility. Login to comment 243 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys Hence, we cannot say that R334C changes its position between open and closed states but rather must limit ourselves to saying that the orientation of R334C relative to the other residue or the distance between them changes as a function of channel gating. Login to comment 244 ABCC7 p.Arg334Cys Nonetheless, these data suggest that changes in the rate coefficients for thiol-modifying reagents at R334C under different experimental conditions reflect conformational changes in the outer vestibule of the CFTR pore, which are associated with ATP-dependent gating. Login to comment 246 ABCC7 p.Arg334Cys As described previously (17), channels formed by WT-CFTR and many pore domain mutants, including R334C-CFTR, exhibit two subconductance states (s1 and s2) as well as the full conductance state (f). Login to comment 248 ABCC7 p.Arg334Cys In R334C-CFTR channels, the most stable conducting state is the s2 state, whereas in WT-CFTR channels, the most stable conducting state is the f state (17). Login to comment 249 ABCC7 p.Lys1250Ala ABCC7 p.Arg334Cys Results from the present study show that when R334C-CFTR channels are locked open by either AMP-PNP or the addition of the K1250A mutation, they are locked into the s2 state. Login to comment 252 ABCC7 p.Lys1250Ala Consistent with this notion, we recently reported that WT-CFTR channels locked open by either AMP-PNP or vanadate (and K1250A-CFTR channels with ATP alone) exhibit a reduced frequency of flickery closures compared with WT-CFTR channels in the presence of ATP alone (27). Login to comment 253 ABCC7 p.Arg334Cys R334C-CFTR channels almost always transition briefly to the f state before closure (see the arrowheads in Fig. 3) (17); the f state may represent an unstable conformation that serves as a transition intermediate between the stable s2 state and the stable c state. Login to comment