ABCMdb

PMID: 23442957

Gao X, Bai Y, Hwang TC
Cysteine scanning of CFTR's first transmembrane segment reveals its plausible roles in gating and permeation.
Biophys J. 2013 Feb 19;104(4):786-97. doi: 10.1016/j.bpj.2012.12.048., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCC7 p.Thr338Cys ABCC7 p.Ala107Cys Interestingly, whole-cell A107C-CFTR currents were very sensitive to changes of bath pH as if the introduced cysteine assumes an altered pKa-like T338C in TM6. Login to comment 11 ABCC7 p.Lys95Cys ABCC7 p.Leu102Cys ABCC7 p.Gln98Cys Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR`s gating cycle. Login to comment 52 ABCC7 p.Leu102Cys (B) Cytoplasmic application of MTSES dramatically reduced ATP-gated L102C/Cysless channel currents in an excised inside-out membrane patch. Login to comment 53 ABCC7 p.Phe87Cys (C) Macroscopic current trace showing a lack of effect of MTSES on F87C/Cysless channels. Login to comment 81 ABCC7 p.Leu102Cys Fig. 1 B shows a representative recording of L102C mutant channels in response to the application of MTSES. Login to comment 82 ABCC7 p.Lys95Cys ABCC7 p.Gln98Cys This observed decrease of macroscopic currents is due to covalent modification of the engineered cysteines by the reagent as the effect persisted even after a complete removal of MTSES. Similar observations were made for K95C- and Q98C-CFTR. Login to comment 83 ABCC7 p.Glu92Cys We could only obtain microscopic current with E92C-CFTR probably due to a poor expression, but the application of MTSES decreased the single-channel amplitude of this construct (see below). Login to comment 85 ABCC7 p.Phe87Cys For instance, in the case of the F87C mutant channel, no obvious changes in the mean current were seen during a 1-min application of MTSES (Fig. 1 C). Login to comment 95 ABCC7 p.Gly103Cys Although we failed to detect any currents from G103C-CFTR, all other seven cysteine-substituted constructs did not respond to cytoplasmic application of MTSES to a significant extent (Fig. 2). Login to comment 101 ABCC7 p.Ile106Cys Fig. 3 A shows a continuous whole-cell recording of I106C-CFTR currents. Login to comment 105 ABCC7 p.Tyr109Cys ABCC7 p.Ala107Cys ABCC7 p.Ala107Cys ABCC7 p.Ile106Cys Further addition of a specific CFTR inhibitor, CFTRinh-172 (39,40), caused a minor decrease of the residual current, indicating a drastic reduction of I106C-CFTR currents by external MTSES. Similar results were obtained for A107C- and Y109C-CFTR except that the magnitude of inhibition for A107C-CFTR is significantly smaller (Fig. 3 C). Login to comment 108 ABCC7 p.Lys95Cys ABCC7 p.Leu102Cys ABCC7 p.Gln98Cys We next tested the accessibility to external MTSES on three positions identified by experiments with inside-out patches, namely K95C, Q98C, and L102C, in the same manner and all three positions turned out nonreactive (data not shown). Login to comment 113 ABCC7 p.Ala107Cys The similarity between TM1 and TM6 was reinforced by the following results with A107C mutant channels. Login to comment 114 ABCC7 p.Ala107Cys When recording whole-cell A107C-CFTR current with a chloride gradient (24 mM internal and 156 mM external [Cl ]), we found that instead of an expected outward rectified I-V curve due to this imposed concentration gradient, the observed I-V relationship shows significant inward rectification (Fig. 4 A). Login to comment 124 ABCC7 p.Ala107Cys We next adopted the similar strategy used in Liu et al. (42) to test if A107C-CFTR can be modulated with baths of different pH. Login to comment 125 ABCC7 p.Ala107Cys In Fig. 4 B, A107C-CFTR channel currents were first activated with forskolin in the bath solution with a pH of 7.4; after the current level stabilized, the bath was switched to forskolin-containing solution with a pH of 6 or 8. Login to comment 128 ABCC7 p.Thr338Cys As shown in Fig. S1 in the Supporting Material, the pKa value of C107 is 7.25, which is nearly identical to that of T338C in TM6 (42). Login to comment 135 ABCC7 p.Ile106Cys (A) A continuous recording of whole-cell I106C/Cysless channel current in response to an external application of MTSES. Login to comment 137 ABCC7 p.Ser108Cys (B) The same protocol was adopted for S108C/Cysless mutant channels, which exhibit little response to external MTSES. Login to comment 139 ABCC7 p.Gly103Cys ABCC7 p.Asp110Cys (C) Summary of whole-cell SCAM results on the eight residues, G103C-D110C. Login to comment 142 ABCC7 p.Gly103Cys * indicates no detectable current for G103C-CFTR. Login to comment 143 ABCC7 p.Ala107Cys FIGURE 4 Effects of external MTSES and pH on A107C-CFTR. Login to comment 144 ABCC7 p.Ala107Cys (A) Whole-cell A107C-CFTR currents in response to external MTSES (left) and I-V curves extracted from the whole-cell recording as marked. Login to comment 145 ABCC7 p.Ala107Cys (B) Effects of acidic or alkaline pH on the whole-cell A107C-CFTR currents (left). Login to comment 149 ABCC7 p.Leu102Cys For instance, for L102C mutant channels, we found that macroscopic currents were increased by 108.5 5 9.5% (n &#bc; 6) after modification by MTSETapplied to the cytoplasmic side of the channel in excised inside-out patches (Fig. 5 A). Login to comment 150 ABCC7 p.Leu102Cys Also similar to what has been reported for positions I344 and M348 in TM6, following MTSET modification of L102C-CFTR, robust activity was observed even in the complete absence of ATP. Login to comment 152 ABCC7 p.Lys95Cys ABCC7 p.Gln98Cys For Q98C and K95C mutant channels, the increases in the mean current amplitude following MTSET modification were ~2- and 6-fold, respectively. Login to comment 153 ABCC7 p.Lys95Cys Because the single-channel conductance was drastically decreased in K95C-CFTR, we were not able to assess the gating effect of MTSET modification. Login to comment 154 ABCC7 p.Gln98Cys Fig. S2, however, shows that MTSET modification of Q98C-CFTR increases both the open probability and the single-channel amplitude. Login to comment 155 ABCC7 p.Leu102Cys The remarkable effects of MTSET on L102C-CFTR currents shown in Fig. 5 A prompted us to examine this effect more closely with single-channel recordings. Login to comment 156 ABCC7 p.Leu102Cys Fig. 5 B shows single-channel traces before and after MTSET modification of L102C-CFTR. Login to comment 159 ABCC7 p.Glu1371Gln ABCC7 p.Leu102Cys Although previous studies have provided evidence that these short-lived closures are ATP independent (43,44) and could result from voltage-dependent block of the pore by large anions from the cytoplasmic side of the channel (45), it is noted that these events are abundantly present in the double mutant L102C/E1371Q at both negative and positive membrane potentials in the absence of ATP (see Fig. S3). Login to comment 164 ABCC7 p.Met348Cys ABCC7 p.Ile344Cys Similar to what we observed for I344C- and M348C-CFTR (16), this robust ATP-independent gating was seen following modification by MTSET but not by MTS-ethylammonium (MTSEA) (Fig. S4). Login to comment 165 ABCC7 p.Lys95Cys ABCC7 p.Leu102Cys ABCC7 p.Leu102Cys ABCC7 p.Gln98Cys ABCC7 p.Gln98Cys ABCC7 p.Glu92Cys State-dependent modification of E92C-, K95C-, Q98C-, and L102C-CFTR The observation that MTSET modification of Q98C and L102C alters CFTR gating suggests that TM1 indeed participates in gating motions of CFTR. Login to comment 171 ABCC7 p.Glu92Cys As this series of experiments requires macroscopic currents, we could not test the E92C mutants, which express poorly (see Discussion for details). Login to comment 172 ABCC7 p.Lys95Cys However, Fig. 6 A shows a representative recording of current response for K95C-CFTR mutants. Login to comment 174 ABCC7 p.Leu102Cys ABCC7 p.Gln98Cys Similar results were obtained from experiments on L102C (Fig. 6 B) and Q98C mutants. Login to comment 175 ABCC7 p.Leu102Cys These data suggest that the modification rate of cysteines on these positions is FIGURE 5 Gating of MTSET-modified L102C/Cysless channel. Login to comment 176 ABCC7 p.Leu102Cys (A) A continuous current recording of L102C/Cysless channels showing that MTSET modification increases the macroscopic current and renders the current ATP-independent. Login to comment 177 ABCC7 p.Leu102Cys (B) Single-channel recording of the L102C/ Cysless-CFTR. Login to comment 196 ABCC7 p.Lys95Cys ABCC7 p.Pro99Cys ABCC7 p.Leu102Cys ABCC7 p.Gln98Cys Third, in the report by Wang et al. (35), K95C, but not Q98C, P99C, or L102C, can react with internal MTSES even before the channel is activated by PKA and ATP, implying a regulated barrier between positions 95 and 98. Login to comment 203 ABCC7 p.Lys95Cys (A) A representative recording for MTSES modification in the absence of ATP for K95C/Cysless channels. Login to comment 204 ABCC7 p.Leu102Cys (B) A real-time recording with a similar protocol shown in (A) for L102C/Cysless channels. Login to comment 206 ABCC7 p.Lys95Cys ABCC7 p.Leu102Cys ABCC7 p.Gln98Cys (C) Summary of the modification rates for K95C, Q98C, and L102C in the presence of ATP (solid squares). Login to comment 250 ABCC7 p.Glu92Cys Although we were not able to measure the modification rate for E92C because of poor expression, in five patches yielding microscopic current, MTSES appears to modify the introduced cysteine when the channel is opened (see Fig. S7). Login to comment 263 ABCC7 p.Pro99Cys ABCC7 p.Ile106Cys ABCC7 p.Ala96Cys (A) Representative single-channel traces for WT/Cysless, A96C/ Cysless, I106C/Cysless, and P99C mutant channels. Login to comment 265 ABCC7 p.Gly103Cys Single star specifies no channel activity was detected for G103C. Login to comment 274 ABCC7 p.Leu102Cys Once we accept the possibility that TM1 undergoes major conformational changes during gating transitions, it is not surprising to see alterations in gating by the mutation itself (e.g., L102C), as well as by subsequent chemical modifications (Fig. 5). Login to comment 275 ABCC7 p.Glu1371Gln ABCC7 p.Leu102Cys It is nevertheless interesting to note that in L102C/E1371Q-CFTR, the gate can open and close repeatedly even when the NBDs are in a dimeric configuration (Fig. S3). Login to comment 276 ABCC7 p.Leu102Cys On the other hand, modification of L102C by MTSET literally renders the channel permanently open even long after ATP is removed and presumably NBDs have been separated. Login to comment