ABCMdb

PMID: 22966014

Jih KY, Sohma Y, Hwang TC
Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation.
J Gen Physiol. 2012 Oct;140(4):347-59. Epub 2012 Sep 10., [PubMed]

Sentences

No. Mutations Sentence Comment

33 ABCC7 p.Arg352Cys When mutating the positively charged arginine at position 352 (located in the sixth transmembrane segment, TM6) to cysteine, the mutant channel (R352C-CFTR) features two distinct open states with unequal conductance (Bai et al., 2010; compare Cui et al., 2008). Login to comment 34 ABCC7 p.Arg352Cys When mutating the positively charged arginine at position 352 (located in the sixth transmembrane segment, TM6) to cysteine, the mutant channel (R352C-CFTR) features two distinct open states with unequal conductance (Bai et al., 2010; compare Cui et al., 2008). Login to comment 43 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser For example, the drastic effect of nonhydrolyzable ATP analogues or mutations (e.g., E1371S or K1250A) that abolish ATP hydrolysis on the open time supports the notion that ATP hydrolysis is coupled to channel Figure 1.ߓ An updated model illustrating the relationship between an opening/closing cycle of the gate and ATP consumption in CFTR` s NBDs. Login to comment 44 ABCC7 p.Lys1250Ala ABCC7 p.Glu1371Ser For example, the drastic effect of nonhydrolyzable ATP analogues or mutations (e.g., E1371S or K1250A) that abolish ATP hydrolysis on the open time supports the notion that ATP hydrolysis is coupled to channel Figure 1. An updated model illustrating the relationship between an opening/closing cycle of the gate and ATP consumption in CFTR` s NBDs. Login to comment 70 ABCC7 p.Arg352Cys Fig. S1 shows the closed-time distribution for R352C-CFTR. Fig. S2 presents dwell-time distributions for O1 and O2 states. Login to comment 71 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe Fig. S1 shows the closed- time distribution for R352C-CFTR. Fig. S2 presents dwell-time distributions for O1 and O2 states. Login to comment 72 ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe Fig. S3 demonstrates the effects of the W401F mutation on the single-channel kinetics of R352C-CFTR. Login to comment 73 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys R E S U L T S Unique pattern of single-channel gating transitions in Cysless/R352C-CFTR During our previous studies in scanning the pore-lining residues in the sixth transmembrane segment of TMDs (TM6), a unique feature of Cysless/R352C-CFTR caught our attentions. Login to comment 74 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys R E S U L T S Unique pattern of single-channel gating transitions in Cysless/R352C-CFTR During our previous studies in scanning the pore-lining residues in the sixth transmembrane segment of TMDs (TM6), a unique feature of Cysless/R352C-CFTR caught our attentions. Login to comment 79 ABCC7 p.Arg352Cys ABCC7 p.Glu1371Ser Consistent with this idea, ATP only induces C࢐O1࢐C transitions in E1371S/R352C-CFTR, a hydrolysis-deficient mutant. Login to comment 80 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Glu1371Ser Consistent with this idea, ATP only induces C→O1→C transitions in E1371S/R352C-CFTR, a hydrolysis-deficient mutant. Login to comment 81 ABCC7 p.Arg352Cys The R352C mutant channel becomes a precious tool, as it allows us to distinguish the prehydrolytic (O1) and post-hydrolytic (O2) open states and thus to "visualize" ATP hydrolysis taking place within each opening burst. Login to comment 103 ABCC7 p.Arg352Cys Figure 2.ߓ Cysless/R352C-CFTR reveals two different open states with distinct conductance level. Login to comment 104 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys Figure 2. Cysless/R352C-CFTR reveals two different open states with distinct conductance level. Login to comment 105 ABCC7 p.Arg352Cys (A) Five representative traces and amplitude histograms from a patch that contained only one Cysless/R352C-CFTR channel. Login to comment 106 ABCC7 p.Arg352Cys (B) Four representative traces and amplitude histograms for Cysless/R352C-CFTR recorded in a condition similar to that in A, except that both pipette and perfusion solution contain 375 mM Cl&#e032; (see Materials and methods for details). Login to comment 107 ABCC7 p.Arg352Cys (B) Four representative traces and amplitude histograms for Cysless/R352C-CFTR recorded in a condition similar to that in A, except that both pipette and perfusion solution contain 375 mM Cl (see Materials and methods for details). Login to comment 111 ABCC7 p.Arg352Cys R352C-CFTR channels is that a higher percentage of C࢐O1࢐O2࢐C transitions were present under the WT background (Table 1). Login to comment 112 ABCC7 p.Arg352Cys R352C-CFTR channels is that a higher percentage of C→O1→O2→C transitions were present under the WT background (Table 1). Login to comment 113 ABCC7 p.Arg352Cys ABCC7 p.Glu1371Ser To further test our hypothesis that the dominant O1࢐O2 transition versus O2࢐O1 transition is the result of ATP hydrolysis, we engineered the E1371S mutation into R352C-CFTR to abolish ATP hydrolysis (Vergani et al., 2003; Bompadre et al., 2005b) and recorded ATP-dependent opening events. Login to comment 114 ABCC7 p.Arg352Cys ABCC7 p.Glu1371Ser To further test our hypothesis that the dominant O1→O2 transition versus O2→O1 transition is the result of ATP hydrolysis, we engineered the E1371S mutation into R352C-CFTR to abolish ATP hydrolysis (Vergani et al., 2003; Bompadre et al., 2005b) and recorded ATP-dependent opening events. Login to comment 115 ABCC7 p.Glu1371Ser This lack of transitions to the O2 state for ATP-induced opening bursts is not caused by the mutation, E1371S, eliminating the O2 state, because in the absence of ATP, the channel opens predominantly into bursts of a larger conductance level corresponding to that of the O2 state (Fig. 3 C). Login to comment 116 ABCC7 p.Glu1371Ser This lack of transitions to the O2 state for ATP-induced opening bursts is not caused by the mutation, E1371S, eliminating the O2 state, because in the absence of ATP, the channel opens predominantly into bursts of a larger conductance level corresponding to that of the O2 state (Fig. 3 C). Login to comment 124 ABCC7 p.Arg352Cys They showed that in addition to O1 and O2 (named s1 and s2, respectively, in Cui et al., 2008), R352C-CFTR occasionally transits to a full conductance level that is not different from that of WT-CFTR. Login to comment 125 ABCC7 p.Arg352Cys They showed that in addition to O1 and O2 (named s1 and s2, respectively, in Cui et al., 2008), R352C-CFTR occasionally transits to a full conductance level that is not different from that of WT-CFTR. Login to comment 129 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ATP hydrolysis drives the O1࢐O2 transition Although our initial observations were made with Cys-less/R352C-CFTR, this unique pattern of gating transitions was also seen when we introduced the R352C mutation into the WT background (Fig. 3 A and Table 1). Login to comment 130 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe ATP hydrolysis drives the O1→O2 transition Although our initial observations were made with Cysless/R352C-CFTR, this unique pattern of gating transitions was also seen when we introduced the R352C mutation into the WT background (Fig. 3 A and Table 1). Login to comment 131 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe One notable difference between R352C- and Cysless/ Ta b l e 1 Summary of opening events by different gating patterns in three CFTR mutants Gating topology O1-O2 O1 O2 O2-O1 (O1-O2) n # Total Cysless/R352C 2.75 mM ATP 100 µM ATP 720 (45%) 663 (56%) 290 (18%) 216 (18%) 175 (11%) 137 (12%) 42 (3%) 32 (3%) 375 (23%) 128 (11%) 1,602 (100%) 1,176 (100%) R352C 2.75 mM ATP 100 µM ATP 834 (55%) 1,246 (59%) 301 (20%) 406 (19%) 173 (11%) 281 (13%) 39 (3%) 45 (2%) 169 (11%) 121 (6%) 1,516 (100%) 2,099 (100%) R352C/W401F 2.75 mM ATP 100 µM ATP 733 (44%) 1,189 (54%) 326 (19%) 367 (17%) 122 (7%) 337 (15%) 28 (2%) 60 (3%) 474 (28%) 232 (11%) 1,683 (100%) 2,185 (100%) Five different gating patterns are illustrated on the top of the table. Login to comment 139 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys Table 1 shows that for both Cysless/R352C and R352C mutant channels, C࢐O1࢐O2࢐C is the prevailing transition; thus, most gating events indeed follow the long-held one-to-one stoichiometry between the gating cycle and ATP hydrolysis cycle. Login to comment 140 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys Table 1 shows that for both Cysless/R352C and R352C mutant channels, C→O1→O2→C is the prevailing transition; thus, most gating events indeed follow the long-held one-to-one stoichiometry between the gating cycle and ATP hydrolysis cycle. Login to comment 141 ABCC7 p.Arg352Cys A simplified scheme (Scheme 1) summarizes the idea that the gating transition pattern observed in R352C-CFTR represents a cyclic steady state in which ATP hydrolysis drives a "clockwise" movement around the state diagram (compare Richard and Miller, 1990; Gunderson and Kopito, 1995). Login to comment 142 ABCC7 p.Arg352Cys A simplified scheme (Scheme 1) summarizes the idea that the gating transition pattern observed in R352C-CFTR represents a cyclic steady state in which ATP hydrolysis drives a "clockwise" movement around the state diagram (compare Richard and Miller, 1990; Gunderson and Kopito, 1995). Login to comment 143 ABCC7 p.Arg352Cys (A) Four representative traces and amplitude histograms show the gating pattern of R352C-CFTR channel in the presence of 2.75 mM ATP. Login to comment 144 ABCC7 p.Arg352Cys (A) Four representative traces and amplitude histograms show the gating pattern of R352C-CFTR channel in the presence of 2.75 mM ATP. Login to comment 145 ABCC7 p.Arg352Cys ABCC7 p.Glu1371Ser (B and C) Representative traces and amplitude histograms for R352C/E1371S-CFTR in the presence (B) or absence (C) of 2.75 mM ATP. Login to comment 146 ABCC7 p.Arg352Cys ABCC7 p.Glu1371Ser (B and C) Representative traces and amplitude histograms for R352C/E1371S-CFTR in the presence (B) or absence (C) of 2.75 mM ATP. Login to comment 149 ABCC7 p.Arg352Cys However, these nondiscernible closures, if they exist, are not the same as the original closed state (C in Scheme 1) for the following reasons: (a) the closed state of R352C-CFTR marked with stars in Fig. 2 assumes a very long lifetime (1 s [Fig. S1] vs. 300-400 ms for WT-CFTR); thus, the probability of having this state with a lifetime of <3 ms buried in an opening burst is extremely small (<0.003); (b) for this idea to be valid, one has to propose that ATP can open this presumed brief closed channel at a rate that is two to three orders faster than it does to the original closed state; and (c) as shown in the next section, multiple rounds of O1→O2 transitions can take place within an opening burst in conditions when the O2 state is stabilized, a theme predicted by Scheme 2. Login to comment 153 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys This idea is first supported simply by comparing the Cysless/R352C- and R352C-CFTR results shown in Table 1. Login to comment 154 ABCC7 p.Arg352Cys The Cysless/R352C mutant channel has an O2 state that is about four times as long as that in that hydrolysis of more than one ATP molecule does take place within an opening burst. Login to comment 163 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys Opening events of Cysless/R352C- (A) or R352C-CFTR (B) containing one (events 1, 3, and 4 in A, and 1-4 in B) or more (events 2 and 5 in A, and 5 in B) O1→O2 transitions. Login to comment 164 ABCC7 p.Ile344Cys Events were extracted from traces in Figs. 2 B and 3 A. Chosen events were specified in boxes and numbered in Figs. 2 B and 3 A. in Cysless/I344C-CFTR, these long ATP-independent openings should show up as the larger O2 state, and that, in the presence of ATP, an opening burst could contain numerous O1→O2 transitions. Login to comment 165 ABCC7 p.Ile344Cys Chosen events were specified in boxes and numbered in Figs. 2 B and 3 A. in Cysless/I344C-CFTR, these long ATP-independent openings should show up as the larger O2 state, and that, in the presence of ATP, an opening burst could contain numerous O1࢐O2 transitions. Login to comment 166 ABCC7 p.Arg352Gln ABCC7 p.Ile344Cys We introduced the R352Q mutation into the Cysless/ I344C-CFTR channel. Login to comment 167 ABCC7 p.Arg352Gln ABCC7 p.Arg352Gln ABCC7 p.Arg352Cys ABCC7 p.Ile344Cys ABCC7 p.Ile344Cys Before MESET modification, Cysless/I344C/R352Q mutant channels behaved similarly as Cysless/R352C-CFTR in the presence of ATP (Fig. 5 A). Login to comment 168 ABCC7 p.Arg352Gln ABCC7 p.Arg352Cys ABCC7 p.Ile344Cys Before MESET modification, Cysless/I344C/R352Q mutant channels behaved similarly as Cysless/R352C-CFTR in the presence of ATP (Fig. 5 A). Login to comment 170 ABCC7 p.Arg352Gln ABCC7 p.Arg352Cys ABCC7 p.Ile344Cys After MTSET modification of Cysless/I344C/R352Q, we indeed observed robust ATP-independent openings (Fig. 5 B) with an open lifetime of 1.03 ± 0.30 s (n = 10), the R352C-CFTR (Fig. S2). Login to comment 171 ABCC7 p.Arg352Gln ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Ile344Cys Correspondingly, the percentage of opening bursts encompassing more than one O1→O2 transition is higher in Cysless/R352C (Table 1). Login to comment 172 ABCC7 p.Arg352Cys Correspondingly, the percentage of opening bursts encompassing more than one O1࢐O2 transition is higher in Cysless/R352C (Table 1). Login to comment 174 ABCC7 p.Ile344Cys In Bai et al. (2010), we showed that after Cysless/I344C-CFTR is modified by MTSET, the open probability of this CFTR mutant in the presence of ATP becomes virtually 1 (Bai et al., 2010). Login to comment 175 ABCC7 p.Ile344Cys In Bai et al. (2010), we showed that after Cysless/I344C-CFTR is modified by MTSET, the open probability of this CFTR mutant in the presence of ATP becomes virtually 1 (Bai et al., 2010). Login to comment 177 ABCC7 p.Arg352Gln ABCC7 p.Ile344Cys Representative traces and amplitude histograms for Cysless/ I344C/R352Q-CFTR under these conditions: (A) in the presence of 2.75 mM ATP; (B-C) after MTSET modification, in the absence (B) or presence (C) of 2.75 mM ATP. Login to comment 178 ABCC7 p.Arg352Gln ABCC7 p.Ile344Cys Representative traces and amplitude histograms for Cysless/ I344C/R352Q-CFTR under these conditions: (A) in the presence of 2.75 mM ATP; (B-C) after MTSET modification, in the absence (B) or presence (C) of 2.75 mM ATP. Login to comment 179 ABCC7 p.Arg352Gln ABCC7 p.Ile344Cys (E) The amplitude of O1 and O2 states of Cysless/I344C/R352Q-CFTR before (the left bar) or after (the right bar) being modified by MTSET. Login to comment 180 ABCC7 p.Arg352Gln ABCC7 p.Ile344Cys (E) The amplitude of O1 and O2 states of Cysless/I344C/R352Q-CFTR before (the left bar) or after (the right bar) being modified by MTSET. Login to comment 181 ABCC7 p.Arg352Cys P < 0.05. with R352C-CFTR (Fig. 3), this double mutant also exhibits a preferred order of the gating transition. Login to comment 182 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe Quantitative analysis of gating events indeed demonstrates a higher percentage of gating events with reentry transitions in W401F/R352C-CFTR (Table 1). Login to comment 183 ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe Quantitative analysis of gating events indeed demonstrates a higher percentage of gating events with reentry transitions in W401F/R352C-CFTR (Table 1). Login to comment 184 ABCC7 p.Arg352Cys The results reveal that the R352C mutation significantly shortens the total open time (150 ms [Fig. S2] vs. 400 ms for WT-CFTR ; Vergani et al., 2003; Bompadre et al., 2005a). Login to comment 185 ABCC7 p.Arg352Cys The results reveal that the R352C mutation significantly shortens the total open time (&#e07a;150 ms [Fig. S2] vs. &#e07a;400 ms for WT-CFTRÊf;; Vergani et al., 2003; Bompadre et al., 2005a). Login to comment 186 ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe Prolonged O1 and O2 dwell times were also found in W401F/R352C-CFTR, but to a lesser extent. Login to comment 187 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe The effect of Cysless and W401F mutations in prolonging the open time of R352C mutant channels is consistent with the observations made for the same mutants in the WT background (Bai et al., 2010; Tsai et al., 2010a). Login to comment 188 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe D I S C U S S I O N An accidental discovery of the R352C mutation grants us the opportunity to actually "see"-in real time-ATP hydrolysis taking place during CFTR gating as the ordered transition between two distinct levels of open channel conductance (O1 and O2) indicates an input of the free energy from ATP hydrolysis. Login to comment 189 ABCC7 p.Arg352Cys D I S C U S S I O N An accidental discovery of the R352C mutation grants us the opportunity to actually "see"-in real time-ATP hydrolysis taking place during CFTR gating as the ordered transition between two distinct levels of open channel conductance (O1 and O2) indicates an input of the free energy from ATP hydrolysis. Login to comment 194 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys After we made our discovery with R352C-CFTR, we recorded WT-CFTR under conditions described in these early reports, but did which is approximately fivefold longer than the mean lifetime of the O2 state for Cysless/R352C-CFTR (Fig. S2). Login to comment 195 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys After we made our discovery with R352C-CFTR, we recorded WT-CFTR under conditions described in these early reports, but did which is approximately fivefold longer than the mean lifetime of the O2 state for Cysless/R352C-CFTR (Fig. S2). Login to comment 206 ABCC7 p.Trp401Phe W401F mutation promotes O2→O1 transition In our latest paper (Jih et al., 2012), using nonhydrolyzable ATP analogues (pyrophosphate or adenylyl-imidodi- phosphate) as baits, we captured a post-hydrolytic state (state X), which, like the O2 state in this paper, can reenter the prehydrolytic open state (O1) upon ATP binding to the vacant ABP2. Login to comment 208 ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe Interestingly, a conservative mutation in NBD1 (W401F, both W and F are aromatic amino acids) can enhance the responsiveness of state X to ATP as well as nonhydrolyzable ATP analogues for reasons yet to be elucidated. Login to comment 209 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe Nevertheless, the striking functional similarities between state X in Jih et al. (2012) and the O2 state in this paper prompt us to test the effect of the W401F mutation on R352C-CFTR. Fig. S3 shows a representative single-channel trace of W401F/R352C-CFTR. Login to comment 210 ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe Compared state C is nearly 1 s for R352C-CFTR. Login to comment 211 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe Nevertheless, the striking functional similarities between state X in Jih et al. (2012) and the O2 state in this paper prompt us to test the effect of the W401F mutation on R352C-CFTR. Fig. S3 shows a representative single-channel trace of W401F/R352C-CFTR. Login to comment 212 ABCC7 p.Arg352Cys Compared state C is nearly 1 s for R352C-CFTR. Login to comment 228 ABCC7 p.Arg352Cys Nonetheless, the mutant R352C does offer the advantage of observing transitions between the O1 and O2 states with a much better temporal resolution necessary for a more thorough microscopic kinetic analysis. Login to comment 230 ABCC7 p.Arg352Cys Nonetheless, the mutant R352C does offer the advantage of observing transitions between the O1 and O2 states with a much better temporal resolution necessary for a more thorough microscopic kinetic analysis. Login to comment 254 ABCC7 p.Thr1246Asn Indeed, mutations of the amino acid residue that directly interacts with ATP have been reported to decrease the opening rate in an ATP-dependent manner (Zhou et al., 2006); further, mutations located at the NBD dimer interface have also been shown to lower the opening rate (e.g., T1246N in Vergani et al., 2005). Login to comment 255 ABCC7 p.Arg352Cys Here, we show that mutating R352 leads to a reduced opening rate (1 s1 for R352C-CFTR vs. 2.5 s1 for WT-CFTR; Vergani et al., 2003; Bompadre et al., 2005a). Login to comment 256 ABCC7 p.Arg352Cys ABCC7 p.Thr1246Asn Figure 7. R352C shorten the locked-open time of hydrolytic-deficient CFTR mutant. Login to comment 257 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn Macroscopic current traces of E1371S-CFTR (A), R352C/E1371S-CFTR (B), T1246N/E1371S-CFTR (C), and R352C/T1246N/E1371S-CFTR (D). Login to comment 258 ABCC7 p.Arg352Cys Figure 7.ߓ R352C shorten the locked-open time of hydrolytic-deficient CFTR mutant. Login to comment 259 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn The current relaxation was fitted with a single-exponential function resulting in the relaxation time constant for each mutant: 65.6 ± 10.1 s (n = 8) for E1371S-CFTR, 4.9 ± 0.8 s (n = 12) for R352C/ E1371S-CFTR, 7.8 ± 1.6 s (n = 7) for T1246N/E1371S-CFTR, and 2.27 ± 0.27 s (n = 6) for R352C/T1246N/E1371S-CFTR. Login to comment 261 ABCC7 p.Arg352Cys ABCC7 p.Arg352Cys ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Glu1371Ser ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn *, P < 0.05 compared with E1371S; #, P < 0.05 between two designated data. Login to comment 263 ABCC7 p.Glu1371Ser *, P < 0.05 compared with E1371S; #, P < 0.05 between two designated data. Login to comment 266 ABCC7 p.Arg352Cys In conclusion, capitalizing on the unique gating features of R352C-CFTR, we were able to visualize ATP hydrolysis in real time for CFTR by simply monitoring single-channel current traces. Login to comment 268 ABCC7 p.Arg352Cys In conclusion, capitalizing on the unique gating features of R352C-CFTR, we were able to visualize ATP hydrolysis in real time for CFTR by simply monitoring single-channel current traces. Login to comment 292 ABCC7 p.Arg352Cys ABCC7 p.Thr1246Asn It follows that mutations that decrease the rate of NBD dimerization (CATP→CAD), e.g., T1246N in Vergani et al. (2005), or those that reduce the rate for CAD→O1 (presumably the R352C mutation), will accelerate the decay rate of macroscopic currents in hydrolysis-deficient mutants upon ATP washout. Login to comment 293 ABCC7 p.Arg352Cys ABCC7 p.Glu1371Ser ABCC7 p.Thr1246Asn Here, in Fig. 7, we show that both R352C and T1246N mutations significantly shorten the locked-open time of the hydrolytic-deficient E1371S-CFTR (Fig. 7). Login to comment 294 ABCC7 p.Arg352Cys ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn ABCC7 p.Lys1250Arg Notably, this result is somewhat different from that shown in Vergani et al. (2005) in which the current relaxation is prolonged in T1246N/K1250R- CFTR. Login to comment 295 ABCC7 p.Arg352Cys ABCC7 p.Glu1371Ser ABCC7 p.Thr1246Asn Here, in Fig. 7, we show that both R352C and T1246N mutations significantly shorten the locked-open time of the hydrolytic-deficient E1371S-CFTR (Fig. 7). Login to comment 296 ABCC7 p.Arg352Cys ABCC7 p.Thr1246Asn ABCC7 p.Thr1246Asn ABCC7 p.Lys1250Arg Nonetheless, the locked-open time is further shortened when combining R352C and T1246N mutations together (Fig. 7, D and E), suggesting that the two mutations affects two different kinetic steps as described above. Login to comment 298 ABCC7 p.Arg352Cys ABCC7 p.Thr1246Asn Nonetheless, the locked-open time is further shortened when combining R352C and T1246N mutations together (Fig. 7, D and E), suggesting that the two mutations affects two different kinetic steps as described above. Login to comment