ABCMdb

ABCC7 p.Arg352Cys

None has been submitted yet.

Publications

PMID: 10220340 [PubMed] Guinamard R et al: "Arg352 is a major determinant of charge selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel."

No. Sentence Comment

101 In cell-attached patches, with 140 mM NaCl in the pipet, the single-channel conductances (in picosiemens) were 6.0 ( 0.3 for the wild type (n ) 7), 5.3 ( 0.3 for R352C (n ) 11), 4.2 ( 0.1 for R352Q (n ) 10), 4.0 ( 0.2 for R352H (n ) 4), and 5.7 ( 0.2 for Q353C (n ) 8). Login to comment
106 In cells expressing the R352C mutation, active channels were observed in about half of the cell-attached patches; 0.6 ( 0.3 (n ) 12 patches) channel was observed before activation, and 4.9 ( 0.8 channels were observed after application of the cAMP-activating reagents. Login to comment
110 Following activation with cAMP, cells transfected with wild-type CFTR had a conductance of 12 ( 2 nS (n ) 6) and cells transfected with R352C had a conductance of 2 ( 0.3 nS (n ) 8) (Figure 2A). Login to comment
113 The single-channel conductances (in picosiemens) were 6.2 ( 0.5 for the wild type (n ) 6)2 (Figure 3B), 5.9 ( 0.3 for R352C (n ) 5), 4.2 ( 0.1 for R352Q (n ) 8), and 5.7 ( 0.3 for Q353C (n ) 8). Login to comment
129 (A) Average whole-cell current-voltage relationships obtained from cells expressing either wild-type CFTR (b) or the R352C mutant (O) in symmetrical Cl--containing solutions. Login to comment
130 Note the much lower level of current in the R352C mutant than in the wild type. Login to comment
131 (B) Average single-channel current-voltage relationships for the wild type (O) and the R352C (0), R352Q (3), and R352H (bath pH of 7.2) (]) mutants. Login to comment
132 The symbols for the wild type and R352C are indistinguishable at several voltages. Login to comment
164 These uncharged substitutions also shifted the reversal potential by an amount comparable to that observed by deprotonating R352H; Erev ) -34.9 ( 2 mV (n ) 7) for R352C, and Erev ) -26.3 ( 1.9 mV (n ) 9) for R352Q (Figure 6), resulting in calculated PCl/PNa ratios of 7 (range of 6-8) and 4 (range of 3.5-4.4), respectively. Login to comment
200 Similar reversal potentials were observed for the R352C mutant: 7 ( 1 mV (n ) 8) for Br- , -13 ( 1 mV (n ) 6) for I- , and -27 ( 2 mV (n ) 5) for F- . Login to comment
209 (A and C) Average single-channel current-voltage relationships for the R352C (A) and R352Q (C) mutants in symmetrical 140 mM NaCl (2) and in the presence of the 10-fold NaCl gradient (O) as described in the legend of Figure 3A. Login to comment
212 (B and D) Single-channel recordings are shown for the R352C (B) and R352Q (D) mutants. Login to comment
217 For the R352C mutant, the conductances were similar to that of the wild type (5.9 ( 0.3 pS for Cl-, 5.2 ( 0.3 pS for Br-, 5.7 ( 0.2 pS for F-, and 3.6 ( 0.2 pS for I-). Login to comment
218 The mechanism for the decreased I- conductance compared to those of the other halide ions for both WT and R352C is not known, although in the R347D mutant the effect is eliminated (2). Login to comment
236 Table 1: Halide Permeability Ratios (PX/PCl)a bromide chloride iodide fluoride wild type 1.39 ( 0.05 1 0.71 ( 0.02 0.25 ( 0.04 R352C 1.04 ( 0.04 1 0.54 ( 0.05 0.22 ( 0.03 a Permeability ratios for the halides relative to those for Cl- for the wild type (WT) and the R352C mutant. Login to comment
248 We do know, however, that Arg352 is in the channel lining because a cysteine substituted for Arg352 reacted with charged, sulfhydryl-specific MTS reagents (19) and the reaction rates were voltage-dependent (21). Login to comment
261 This is unlikely to be the mechanism in CFTR because Arg352 does not appear to form a high-affinity Cl-binding site because the mutation R352C does not alter the halide selectivity sequence. Login to comment

PMID: 11585852 [PubMed] Smith SS et al: "CFTR: covalent and noncovalent modification suggests a role for fixed charges in anion conduction."

No. Sentence Comment

25 R347C and R352C were gifts of M. Akabas (Albert Einstein College of Medicine, Bronx, NY) and used in their parent vector, pMN. Login to comment
107 The Function of R334C and K335C CFTR Was Modified by External MTSES or MTSET but the Function of R347C and R352C CFTR Was Not Modified by these Polar Thiol Reagents Fig. 3 summarizes the results of experiments in which MTSES, MTSET, or MTSEA (100 ␮M-10 mM) were added to the solution bathing oocytes expressing wt, R334C, K335C, R347C, or R352C CFTR. Login to comment
129 In the case of R352C CFTR, our observations also differed substantially from those reported by Cheung and Akabas (1996, 1997). Login to comment
131 In contrast, we were not able to detect effects of this magnitude applying either MTSES or MTSET to oocytes expressing R352C CFTR (Fig. 5 A). Login to comment
133 Comparison of the effects of MTSES, MTSET, and MTSEA on the conductance of oocytes expressing R334C, K335C, R347C, or R352C CFTR. Login to comment
140 MTSET and MTSES, polar thiol reagents, did not produce a discernible alteration in conductance of oocytes expressing R352C CFTR. Login to comment
144 (B) Addition of 1 mM MTSEA produced a sixfold increase in conductance of an oocyte expressing R352C CFTR not previously exposed to MTSET. Login to comment
151 R352C CFTR Function Is Modified by Addition of 1 mM MTSEA to the Perfusate, However the Effect Is Not Cysteine-specific Shown in Fig. 5 B is the result of a representative exper- iment in which an oocyte expressing R352C CFTR was exposed to 1 mM MTSEA; the result being a sixfold increase in conductance. Login to comment
186 In view of the results obtained with R352C CFTR, we tested for effects of exposure to MTSET and MTSES using constructs bearing substitutions other than cysteine for R334. Login to comment
435 Even if we allow for the possibility of some modest accessibility of R352C, it is evident that the outer sites are highly reactive, whereas the inner sites are either not at all reactive or much less so. Login to comment
442 This is an important point because in the course of our experiments (e.g., R352C CFTR) and those of others using cysteine accessibility or pH titration (Coulter et al., 1995; Akabas, 1998), it has become apparent that structural changes can render sites "distant" from the mutation accessible or titratable. Login to comment

PMID: 17043152 [PubMed] Aubin CN et al: "Positive charges at the intracellular mouth of the pore regulate anion conduction in the CFTR chloride channel."

No. Sentence Comment

112 Unfortunately, R352C Figure 6. Login to comment
181 to poor expression of the R352C mutant, and as a result it remains uncertain whether this charged amino acid contributes directly to pore surface charge. Login to comment

PMID: 18056267 [PubMed] Beck EJ et al: "Conformational changes in a pore-lining helix coupled to cystic fibrosis transmembrane conductance regulator channel gating."

No. Sentence Comment

218 Finally, the MTSEA reactivity was restricted to only five of twenty-six residues in and flanking TM6 in our study, whereas in the earlier study, residues F337C, S341C, I344C, R347C, T351C, R352C, and Q353C were also shown to be accessible to MTS reagents. Login to comment

PMID: 18421494 [PubMed] Cui G et al: "Mutations at arginine 352 alter the pore architecture of CFTR."

No. Sentence Comment

98 Previous studies showed that both R347C and R352C either were not accessible to membrane-impermeant MTS reagents (methanethiosulfonate ethyltrimethylammonium [MTSET+ ] or methanethiosulfonate ethylsulfonate [MTSES- ]) applied to the extracellular solution or lacked significant functional consequences when modified; this suggested that both sites either were at the predicted cytoplasmic end of the pore, and therefore cytoplasmic to the narrow region, or were not pore-facing residues (Smith et al. 2001). Login to comment
99 R352C was, however, sensitive to membrane-permeant MTSEA+ . Login to comment
229 We previously reported that a cysteine engineered at R352 either was not accessible to the membrane-impermeable reagents MTSES- and MTSET+ applied externally or lacked significant functional consequences when modified, although R352C-CFTR (but not WT-CFTR) did respond to prolonged exposure to the membrane-permeant MTSEA+ (Smith et al. 2001). Login to comment
230 Surprisingly, similar results were found in R352Q-CFTR, suggesting that the response to MTSEA+ in R352C-CFTR was nonspecific, not being due to modification of that engineered cysteine. Login to comment
263 Designation of R352 as the anion selectivity filter was predominantly based upon differences in the rates of modification of engineered cysteines at this site (in R352C-CFTR) by positively and negatively charged sulfhydryl modifying reagents, MTSET+ and MTSES- (Cheung and Akabas 1997; Guinamard and Akabas 1999). Login to comment

PMID: 19754156 [PubMed] Alexander C et al: "Cystic fibrosis transmembrane conductance regulator: using differential reactivity toward channel-permeant and channel-impermeant thiol-reactive probes to test a molecular model for the pore."

No. Sentence Comment

52 We proposed that these spontaneous changes, that are not seen in either wt or Cys-less CFTR, reflect the coordination of trace Table 1: Percent Change in Oocyte Conductance in the Presence of Compounda MTSETþ MTSES- [Ag(CN)2]- [Au(CN)2]- G330C O O O O I331C -51.6 ( 6.3 -28.9 ( 2.1 -63.1 ( 8.8 O I332C O O O O L333C -58.5 ( 4.8 -47.5 ( 7.6 -83.1 ( 2.2 O R334C þ76.9 ( 11.3 -84.4 ( 1.5 -67.4 ( 7.4 -41.4 ( 3.1 K335C þ10.7 ( 2.4 -37.3 ( 1.5 -29.1 ( 6.4 -54.6 ( 4.7 I336C -54.4 ( 7.9 -75.0 ( 0.6 -81.2 ( 10.5 O F337C O O -89.6 ( 1.9 -90.1 ( 1.3 T338C -37.1 ( 3.3 -85.4 ( 2.5 -75.0 ( 5.2 -88.3 ( 1.6 T339C O O -24.5 ( 7.2 O I340C O O -93.8 ( 1.0 O S341C O O -49.3 ( 4.8 O F342C O O -84.7 ( 1.8 O C343 O O O O I344C O O -66.9 ( 9.3 -77.9 ( 2.1 V345C O O -49.1 ( 9.3 O L346C O O O O R347C O O O O M348C O O -47.9 ( 8.8 -50.1 ( 3.3 A349C O O -19.0 ( 2.0 O V350C O O O O T351C O O O O R352C O O -77.5 ( 1.3 O Q353C O O -72.6 ( 4.5 -76.7 ( 2.8 a Values are means ( SE of three or more oocytes. Login to comment
180 We had previously reported that R352C/wt CFTR was not reactive toward MTSETþ and MTSES- (7), a result consistent with the findings of Beck et al. (9). Login to comment
181 We also reported, however, that R352C/wt CFTR displayed an unusual, spontaneously reversible reaction with MTSEAþ (7). Login to comment
182 In those experiments, however, qualitatively identical, reversible reactivity toward MTSEAþ was also seen using either R352Q/wt or R352H/wt CFTR constructs, suggesting that the target of MTSEAþ was 1 of the 18 endogenous cysteines in the R352C/wt protein (7). Login to comment
184 The experiment depicted in Figure 5 demonstrates that R352C/Cys-less CFTR exhibited reactivity toward [Ag(CN)2]- . Login to comment
185 Reactivity toward [Au(CN)2]- was not seen, and experiments with R352C/ wt CFTR yielded identical results (not shown). Login to comment
186 We also confirmed that neither R352C/wt nor R352C/Cys-less exhibits reactivity toward either MTSETþ or MTSES- (not shown), contrary to the reports of Chueng and Akabas (5, 6). Login to comment
212 FIGURE 5: R352C/Cys-less CFTR was reactive toward [Ag(CN)2]- (1 mM) as judged by reversal of inhibition by adding KCN (1 mM) in the continued presence of [Ag(CN)2]- . Login to comment
241 R352C/wt CFTR is not reactive toward MTSETþ and MTSES- , but St. Aubin and Linsdell (40), Cui et al. (41), and Jordan et al. (3) identified R352 as critical for the maintenance of normal anion conduction and gating in CFTR channels. Login to comment
281 Note the lack of consistent results reported for F337C, S341C, I344C, R347C, T351C, R352C, and Q353C (shaded). Login to comment
299 The reactivity of R352C CFTR toward the channel-permeant probe, [Ag(CN)2]- , clearly implicates R352 as a "pore-lining" residue, as suggested by the effect of amino acid substitutions at this position on single-channel conductance, envisioned by St. Aubin and Linsdell (40) as contributing a positive electrostatic potential to a cytoplasmic vestibule for the pore. Login to comment

PMID: 20805575 [PubMed] Bai Y et al: "Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation."

No. Sentence Comment

18 For R352C, which exhibited reduced single-channel amplitude, modifications by two positively charged reagents with different chemical properties completely restored the single-channel amplitude but had distinct effects on both the open time and the closed time. Login to comment
82 7 out of the 25 mutant channels exhibited a reduced single-channel current amplitude, including, from extracellular to intracellular, R334C, K335C, F337C, T338C, S341C, R347C, and R352C (Fig. 2). Login to comment
83 The single-channel amplitude is unsolv- able in the cases of R334C, S341C, R347C, and R352C due to a limited bandwidth, whereas it is 0.2-0.3 pA for Data analysis Current traces containing fewer than three channel opening levels and lasting for >1 min were selected for single-channel kinetic analysis using a program developed by L. Csanády (2000). Login to comment
144 Fig. 7 depicts a representative recording of a patch containing a single cysless/R352C CFTR channel. Login to comment
162 The restoration of the single-channel amplitude by MTSET or MTSEA on R352C compelled us to use another strategy to assess the function of the CFTR pore before and after modification. Login to comment
163 Fig. 8 shows results obtained from patches yielding macroscopic cysless/R352C channel currents. Login to comment
167 When the membrane voltage was held at 50 mV, 50 µM glibenclamide induced 27.2 ± 1.7% (n = 10) block of the Cl current for cysless/R352C. Login to comment
171 However, when R352C was modified by MTSET, the single-channel amplitude was restored to that of the cysless/WT channel (Fig. 7 A). Login to comment
173 Interestingly, although bringing back the positive charge at this position with MTSET completely restores the single-channel amplitude, gating of MTSET-modified cysless/ R352C is not fully recovered to the level of cysless/WT channels. Login to comment
185 Because anion conduction was severely perturbed by the mutations R352C and S341C, we were not able to assess the effects of MTSES modification on the single-channel amplitude for these two constructs. Login to comment
190 For the positions 344, 345, and 348, however, single-channel recordings are not helpful because no visible current glibenclamide at the same holding potential induced stronger block in the cysless/WT channel (48.6 ± 3.0%; n = 5; Fig. 8 D), the effect of the blocker was attenuated by the R352C mutation. Login to comment
197 (A; top trace) A continuous recording showing the effects of MTSET or MTSEA on a single cysless/R352C channel. Login to comment
201 (B) Single-channel amplitude, Po, open time and closed time of MTSET- (blue) and MTSEA-modified (green) cysless/R352C channel, as determined by Gaussian fitting and kinetics analysis; n = 6. inhibition of the macroscopic mean current (Fig. 4 C) and the single-channel current in the case of the cysless/ M348C channel might be due to oxidation of the introduced cysteine to a state not reactive toward either DTT or MTS reagents. Login to comment
214 Although the exact reason is unknown, the discrepancy between the extent of Figure 8. Blocking of cysless/ R352C channels by glibenclamide before and after MTS modification. Login to comment
217 (D) The fraction of block, calculated as (1Ig/ I0) × 100% (Ig and I0 are the mean current in the presence of ATP and ATP plus glibenclamide, respectively), for cysless/WT channels (gray), cysless/R352C channels before modification (black) and after modification with MTSET (blue), and after modification with MTSEA (green). Login to comment
235 The reason for this slow reaction rate is unclear, but it could be due to a perturbation of the pore architecture by the R352C mutation (Cui et al., 2008). Login to comment
302 Second, the potency to an open-channel blocker, glibenclamide, is reduced by the charge-neutralizing mutation R352C (also see Cui et al., 2008), but was restored by charge-restoring adducts. Login to comment

PMID: 9922375 [PubMed] Sheppard DN et al: "Structure and function of the CFTR chloride channel."

No. Sentence Comment

145 The voltage dependence of the reaction rates of MTS reagents with CFTR (Br0 Å Cl0 ú I0 ), Xenopus CFTR (Br0 Å I0 ú Cl0 ), and human-Xenopus CFTR chimeras in which eithercysteine mutations at these residues and single-channel studies of wild-type CFTR and the mutant R352C suggest MSD1 or MSD2 of human CFTR was replaced with the equivalent region of Xenopus CFTR (hX1-6, Br0 Å I0 úthat the selectivity of CFTR for anions over cations is determined by a site located at the intracellular end of the pore Cl0 , and hX7-12, Br0 ú Cl0 ú I0 , respectively) also suggest that sequences in MSD1 likely determine the anionthat involves the residue R352 (32, 52). Login to comment
148 Therefore, other sequences must account for the differ-end of the pore and R352C is located closer to the extracellular end of the pore than either T351C or Q353C (32). Login to comment

PMID: 9922376 [PubMed] Dawson DC et al: "CFTR: mechanism of anion conduction."

No. Sentence Comment

480 Three residues, R334C, R347C, neered cysteines can react with the charged MTS reagents in the pore interior. The potential flaw in this assumptionand R352C, were also inhibited by the larger cation MTSET0 . Login to comment

PMID: 22352759 [PubMed] Norimatsu Y et al: "Cystic fibrosis transmembrane conductance regulator: a molecular model defines the architecture of the anion conduction path and locates a "bottleneck" in the pore."

No. Sentence Comment

345 They found that R352C CFTR exhibited unstable gating and reduced current excursions reminiscent of the subconductance states reported by Cui et al.11 Modification of R352C CFTR by either MTSET+ or MTSEA+ restored the full conductance state and appeared to stabilize the gating pattern. Login to comment

PMID: 22966014 [PubMed] Jih KY et al: "Nonintegral stoichiometry in CFTR gating revealed by a pore-lining mutation."

No. Sentence Comment

34 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
71 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 Fig. S3 demonstrates the effects of the W401F mutation on the single-channel kinetics of R352C-CFTR. Login to comment
74 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
80 Consistent with this idea, ATP only induces C→O1→C transitions in E1371S/R352C-CFTR, a hydrolysis-deficient mutant. Login to comment
81 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
104 Figure 2. Cysless/R352C-CFTR reveals two different open states with distinct conductance level. Login to comment
105 (A) Five representative traces and amplitude histograms from a patch that contained only one Cysless/R352C-CFTR channel. Login to comment
107 (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
112 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
114 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
125 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
130 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 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
140 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
142 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
144 (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
146 (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 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 This idea is first supported simply by comparing the Cysless/R352C- and R352C-CFTR results shown in Table 1. Login to comment
154 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 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
167 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 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 Correspondingly, the percentage of opening bursts encompassing more than one O1→O2 transition is higher in Cysless/R352C (Table 1). Login to comment
181 P < 0.05. with R352C-CFTR (Fig. 3), this double mutant also exhibits a preferred order of the gating transition. Login to comment
182 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 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
186 Prolonged O1 and O2 dwell times were also found in W401F/R352C-CFTR, but to a lesser extent. Login to comment
187 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 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 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
209 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 Compared state C is nearly 1 s for R352C-CFTR. Login to comment
228 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
255 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 Figure 7. R352C shorten the locked-open time of hydrolytic-deficient CFTR mutant. Login to comment
257 Macroscopic current traces of E1371S-CFTR (A), R352C/E1371S-CFTR (B), T1246N/E1371S-CFTR (C), and R352C/T1246N/E1371S-CFTR (D). Login to comment
259 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 *, P < 0.05 compared with E1371S; #, P < 0.05 between two designated data. Login to comment
266 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 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 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 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
296 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
33 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
70 Fig. S1 shows the closed-time distribution for R352C-CFTR. Fig. S2 presents dwell-time distributions for O1 and O2 states. Login to comment
73 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 Consistent with this idea, ATP only induces C࢐O1࢐C transitions in E1371S/R352C-CFTR, a hydrolysis-deficient mutant. Login to comment
103 Figure 2.ߓ Cysless/R352C-CFTR reveals two different open states with distinct conductance level. Login to comment
106 (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
111 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 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
124 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 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
139 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 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 (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 (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
168 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
172 Correspondingly, the percentage of opening bursts encompassing more than one O1࢐O2 transition is higher in Cysless/R352C (Table 1). Login to comment
183 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
185 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
189 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
195 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
211 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 Compared state C is nearly 1 s for R352C-CFTR. Login to comment
230 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
258 Figure 7.ߓ R352C shorten the locked-open time of hydrolytic-deficient CFTR mutant. Login to comment
268 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
295 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
298 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

PMID: 22966013 [PubMed] Tsai MF et al: "CFTR: An ion channel with a transporter-type energy-coupling mechanism."

No. Sentence Comment

27 By mutating residue R352 in the transmembrane domain to cysteine, they created a mutant CFTR channel (R352C-CFTR) that harbors experimentally distinguishable O1 and O2 states not seen in wild-type (WT) channel gating. Login to comment
34 A critical issue that has not been addressed by Jih et al. (2012) is to what degree the R352C mutation distorts normal molecular behaviors of CFTR. Login to comment
37 This allows a qualitative comparison of WT and R352C gating behavior. Login to comment
40 Now, the rate constants derived from recording the R352C mutant could provide constraints for the ML method, which can then be applied to examine CFTR kinetic models in an even more satisfactory detail. Login to comment
50 The authors then took the story one step further by introducing a catalysis-abolishing mutation E1371S into R352C-CFTR; these hydrolysis-deficient channels now open and close reversibly (C→O1→C and C→O2→C), indicating that ATP hydrolysis underlies a unidirectional transition from O1 to O2 (Fig. 1 B). Login to comment
58 By using the R352C mutation as a tool, now Jih et al. (2012) have the ability to directly quantify the rate constants connecting C, O1, and O2 states, and thus better characterize previously undissectible molecular events, such as ATP hydrolysis (O1→O2), at the single molecule level. Login to comment
62 (C) A single-channel event of R352C-CFTR with three conductance states, acquired with my high-resolution word editor. Login to comment
25 By mutating residue R352 in the transmembrane domain to cysteine, they created a mutant CFTR channel (R352C-CFTR) that harbors experimentally distinguishable O1 and O2 states not seen in wild-type (WT) channel gating. Login to comment
32 A critical issue that has not been addressed by Jih et al. (2012) is to what degree the R352C mutation distorts normal molecular behaviors of CFTR. Login to comment
35 This allows a qualitative comparison of WT and R352C gating behavior. Login to comment
38 Now, the rate constants derived from recording the R352C mutant could provide constraints for the ML method, which can then be applied to examine CFTR kinetic models in an even more satisfactory detail. Login to comment
48 The authors then took the story one step further by introducing a catalysis-abolishing mutation E1371S into R352C-CFTR; these hydrolysis-deficient channels now open and close reversibly (C࢐O1࢐C and C࢐O2࢐C), indicating that ATP hydrolysis underlies a unidirectional transition from O1 to O2 (Fig. 1 B). Login to comment
56 By using the R352C mutation as a tool, now Jih et al. (2012) have the ability to directly quantify the rate constants connecting C, O1, and O2 states, and thus better characterize previously undissectible molecular events, such as ATP hydrolysis (O1࢐O2), at the single molecule level. Login to comment
60 (C) A single-channel event of R352C-CFTR with three conductance states, acquired with my high-resolution word editor. Login to comment

PMID: 22923500 [PubMed] Norimatsu Y et al: "Locating a Plausible Binding Site for an Open Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator."

No. Sentence Comment

161 This result is most likely a reflection of an increase in the open probability of I1131C CFTR channels similar to that observed for R352C CFTR by Bai et al., (2010). Login to comment
244 This result is most likely a reflection of an increase in the open probability of I1131C CFTR channels, similar to that observed for the R352C CFTR (Bai et al., 2010). Login to comment

PMID: 9511930 [PubMed] Akabas MH et al: "Probing the structural and functional domains of the CFTR chloride channel."

No. Sentence Comment

128 Thus, when cysteine is substituted for Arg352 the charge selectivity drops to the same low level that is observed in the more extracellular portion of the channel (Fig. 3B). Login to comment
129 If other residues in this region were the main determinants of anion selectivity, then, the anion selectivity of the R352C mutant should have been similar to that of the adjacent residues. Login to comment
130 Moreover, based on our measurements of electrical distance, R352C is closer to the extracellular end of the channel than is T351C or Q353C (Fig. 3A). Login to comment

PMID: 9089437 [PubMed] Cheung M et al: "Locating the anion-selectivity filter of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel."

No. Sentence Comment

9 Furthermore, the electrical distance calculations indicate that R352C is closer to the extracellular end of the channel than either of the adjacent residues. We speculate that the cytoplasmic end of the M6 segment may loop back into the channel narrowing the lumen and thereby forming both the major resistance to current flow and the anion-selectivity filter. Login to comment
107 We did not measure the reaction rate constants for the most extracellular residue, I331C, because we thought that it was unlikely that the reaction rates would be voltage dependent given the absence of voltage dependence at the adjacent, more cytoplasmic residues. We also did not measure the reaction rate constants for the mutants I344C and R347C because, although MTSEAϩ reacted with these residues, MTSES- and MTSETϩ did not react with these k ψ( )( )ln k Ψ 0=( )( ) zFδ RT/( )-ln ψ= t a b l e i Second-order Rate Constants for the Reaction of the MTS Reagents with the Water-exposed Cysteine Mutants k ES (M-1s-1) k EA (M-1s-1) k ET (M-1s-1) mutant -25 mV -50 mV -75 mV -25 mV -50 mV -75 mV -25 mV -50 mV -75 mV L333C 71 Ϯ 3(3) 71 Ϯ 20(2) 71 Ϯ 23(3) 320 Ϯ 89(2) 320 Ϯ 128(2) 333 Ϯ 139(3) 952 Ϯ 136(2) 1,000 Ϯ 350(2) 1,053 Ϯ 443(2) R334C 48 Ϯ 14(2) 48 Ϯ 6(3) 44 Ϯ 8(4) 145 Ϯ 32(2) 163 Ϯ 7(2) 182 Ϯ 21(3) 444 Ϯ 49(2) 454 Ϯ 124(2) 588 Ϯ 95(3) K335C 36 Ϯ 20(3) 23 Ϯ 11(3) 27 Ϯ 16(3) 222 Ϯ 80(3) 121 Ϯ 51(4) 107 Ϯ 30(3) 217 Ϯ 111(3) 235 Ϯ 28(3) 217 Ϯ 95(4) F337C 91 Ϯ 17(2) 80 Ϯ 22(3) 71 Ϯ 20(4) 222 Ϯ 74(2) 222 Ϯ 86(3) 285 Ϯ 81(3) 740 Ϯ 246(3) 740 Ϯ 82(2) 714 Ϯ 51(2) S341C 56 Ϯ 18(3) 56 Ϯ 40(2) 43 Ϯ 12(3) 93 Ϯ 6(3) 110 Ϯ 22(3) 138 Ϯ 34(3) 690 Ϯ 356(3) 556 Ϯ 246(3) 800 Ϯ 224(4) T351C 100 Ϯ 25(5) 57 Ϯ 6(3) 26 Ϯ 9(6) 146 Ϯ 30(4) 195 Ϯ 42(4) 296 Ϯ 18(3) 308 Ϯ 47(10) 392 Ϯ 78(6) 769 Ϯ 89(5) R352C 42 Ϯ 4(3) 26 Ϯ 4(5) 21 Ϯ 6(4) 105 Ϯ 76(3) 137 Ϯ 46(3) 205 Ϯ 58(2) 417 Ϯ 138(4) 800 Ϯ 128(2) 952 Ϯ 408(2) Q353C 125 Ϯ 23(4) 51 Ϯ 12(4) 42 Ϯ 8(4) 83 Ϯ 24(4) 116 Ϯ 42(4) 160 Ϯ 92(3) 189 Ϯ 48(6) 220 Ϯ 48(3) 625 Ϯ 273(4) residues and therefore we could not determine the charge selectivity at these positions.2 The reaction rate constants that we have measured are between 10-and 500-fold slower than the rates of reaction with sulfhydryls in free solution (Table II) (Stauffer and Karlin, 1994). Login to comment
134 Note that the electrical distance to the residues from L333C to S341C is close to zero and that the electrical distance to R352C is smaller than the electrical distance to the adjacent residues. Login to comment
173 We now show that based on the measured electrical distances R352C appears to be closer to the extracellular end of the channel than either of the adjacent residues. Login to comment
186 If other residues in this region were the main determinants of anion selectivity, then, the anion selectivity of the R352C mutant should have been similar to that of the adjacent residues. Login to comment
187 Based on our measurements of electrical distance, R352C is closer to the extracellular end of the channel than T351C and Q353C (Fig. 4, see below). Login to comment
192 A further suggestion that Arg352 is important in charge selectivity is the increase in the reaction rate of the cationic MTSETϩ with the R352C mutant as compared to the adjacent residues (Table II, column 3). Login to comment
193 Removing the positive charge in the R352C mutant may increase the ability of cations to enter this region near the cytoplasmic end of the channel, thereby accounting for the increase rate of reaction of MTSETϩ at R352C compared to the adjacent residues. Login to comment
234 The electrical distances from the extracellular end of the channel to these three residues, with T351C being more cytoplasmic than R352C, is also inconsistent with an ␣-helical secondary structure (Fig. 4). Login to comment

PMID: 8930836 [PubMed] Linsdell P et al: "Disulphonic stilbene block of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in a mammalian cell line and its regulation by a critical pore residue."

No. Sentence Comment

102 Since MTSET reacts covalently with cysteine residues, a very low rate of MTSET permeation would presumably be sufficient to block cysteine-substitutecl forms of CFTR such as R347C and R352C, whereas a similarly low rate of permeation byv a reversible blocker such as gluconate may not affect Cl- permeation. Login to comment

PMID: 8744306 [PubMed] Cheung M et al: "Identification of cystic fibrosis transmembrane conductance regulator channel-lining residues in and flanking the M6 membrane-spanning segment."

No. Sentence Comment

91 Effects of MTS reagents on wild-type cysteines RESULTS in CFTR To identify the residues in and flanking the M6 membrane-spanning segment that are on the water-exposed surface of As reported previously (Akabas et al., 1994b), extracellular applications of the MTS reagents to Xenopus oocytes ex- L2j K329C L. _J *G330C 1331C 1332C L333C R334C K335C 1336C F337C T338C T339C 1340C S341C T342C C343,WT 1344C V345C L346C R347C M348C A349C V350C T351C R352C Q353C 0 2000 4000 6000 8000 0 25 50 PEAK CURRENTS (nA) TIME TO REACH PLATEAU (min) FIGURE 2 Peak CFTR-induced currents and time to reach the plateau current after stimulation with cAMP-activating reagents for 24 cysteine-substitution mutants and wild-type CFTR. Login to comment
109 Accessibility of substituted cysteines to MTSES- A 1-min application of 10 mM MTSES- significantly inhibited the CFIR-induced currents of 9 of the 24 cysteine-substituted mutants (Fig. 4 A), L333C, R334C, K335C, F337C, S341C, R347C, T351C, R352C, and Q353C. Login to comment
116 We also examined the ability of a larger, permanently positively charged reagent, MTSET+, to react with three of the mutants, R334C, R347C, and R352C, that were susceptible to MTSEA+. Login to comment
117 One-minute and 8-min applications of 1 mM MTSET+ inhibited the CFTIR-mediated current of the mutants R334C by 53 ± 6% and 52 ± 7% (n = 3); R347C by 44 ± 2% and 36 + 3% (n = 3) (Fig. 3 D); and R352C by 46 ± 11% and 54.6 ± 10.5% (n = 3). Login to comment
173 Diameter of the channel lumen The ability of MTSET+ to react with the R352C mutant indicates that it can penetrate from the extracellular end to this level. Login to comment
175 Therefore, the channel diameter from the extracellular enid to the position of R352C, near the cytoplasmic end of the M6 segment, must be at least 6 A. Login to comment
90 Effects of MTS reagents on wild-type cysteines RESULTS in CFTR To identify the residues in and flanking the M6 membrane-spanning segment that are on the water-exposed surface of As reported previously (Akabas et al., 1994b), extracellular applications of the MTS reagents to Xenopus oocytes ex- L2j K329C L. _J *G330C 1331C 1332C L333C R334C K335C 1336C F337C T338C T339C 1340C S341C T342C C343,WT 1344C V345C L346C R347C M348C A349C V350C T351C R352C Q353C 0 2000 4000 6000 8000 0 25 50 PEAK CURRENTS (nA) TIME TO REACH PLATEAU (min) FIGURE 2 Peak CFTR-induced currents and time to reach the plateau current after stimulation with cAMP-activating reagents for 24 cysteine-substitution mutants and wild-type CFTR. Login to comment
108 Accessibility of substituted cysteines to MTSES- A 1-min application of 10 mM MTSES- significantly inhibited the CFIR-induced currents of 9 of the 24 cysteine-substituted mutants (Fig. 4 A), L333C, R334C, K335C, F337C, S341C, R347C, T351C, R352C, and Q353C. Login to comment
115 We also examined the ability of a larger, permanently positively charged reagent, MTSET+, to react with three of the mutants, R334C, R347C, and R352C, that were susceptible to MTSEA+. Login to comment
171 Diameter of the channel lumen The ability of MTSET+ to react with the R352C mutant indicates that it can penetrate from the extracellular end to this level. Login to comment

PMID: 23223629 [PubMed] Jih KY et al: "Nonequilibrium gating of CFTR on an equilibrium theme."

No. Sentence Comment

155 Fortuitously, this hydrolysis-dependent O1 &#a1; O2 transition was discerned in a CFTR mutant, R352C. Login to comment
156 Single-channel recordings of R352C-CFTR revealed two distinguishable conductance states: a smaller one (O1), about one-third of the WT conductance, and a larger one (O2), about one-half of the WT conductance (8). Login to comment
161 The idea that the C &#a1; O1 &#a1; O2 &#a1; C preferred transition is driven by ATP hydrolysis is supported not only by the time asymmetry but also by the observation that the O1 &#a1; O2 transition seen in an opening burst is abolished when the hydrolysis-deficient mutation (E1371S) is engineered into the R352C background. Login to comment
162 An even more intriguing observation made with the R352C mutant channel is that, occasionally, there are opening events that consist of more than one O1 &#a1; O2 transition (boxed in FIGURE 3). Login to comment
177 Unique gating features of R352C-CFTR Five representative traces from patches that contain only one R352C-CFTR in the presence of 2.75 mM ATP. Login to comment
178 R352C-CFTR shows two distinct levels of open-state conductance: the smaller O1 and the larger O2. Login to comment
196 The data on R352C-CFTR support the notion that a partial separation of the NBD dimer does not necessarily close the gate in the TMDs, but the fact that the O1 and O2 states exhibit different single-channel amplitudes indicates that ATP hydrolysis and/or subsequent partial separation of NBDs must affect the conformation of the ion permeation pathway in TMDs. Login to comment

PMID: 23440202 [PubMed] Jih KY et al: "Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle."

No. Sentence Comment

6 We further examined the effect of Vx-770 on R352C-CFTR, a unique mutant that allows direct observation of hydrolysis-triggered gating events. Login to comment
40 11 www.pnas.org/cgi/doi/10.1073/pnas.1215982110 finding of a mutant, R352C-CFTR, which allows quantification of ATP hydrolysis-triggered open-to-open transition, imparts a straightforward approach to examine how Vx-770 affects the reentry pathway. Login to comment
41 Our data with R352C-CFTR not only support the hypothesis that Vx-770 lengthens the open time of WT-CFTR by increasing the frequency of reentry, but also spawn unique targets for CFTR potentiators that could complement the action of Vx-770, a crucial step toward the ultimate goal of curing CF. Login to comment
105 Effects of Vx-770 on R352C-CFTR. Login to comment
109 Fortunately, our recent discovery of a CFTR mutant, R352C, that exhibits different single-channel conductance for the O1 and O2 states grants us a unique opportunity to directly assess the effects of Vx-770 on the lifetime of the O2 state. Login to comment
122 report (24), most of the opening bursts in R352C-CFTR contain one O1 ࢐ O2 transition; only ~10% of the openings are categorized as events with more than one O1 ࢐ O2 transition. Login to comment
141 Furthermore, by examining the effects of Vx-770 on R352C-CFTR, we were able to show that this compound indeed increases the frequency of reentry events (Fig. 4 and Table 1). Login to comment
142 Notably, the reentry pathway was first suggested by a report using PPi as a tool to fish out a unique posthydrolytic state (36) and later established by scrutinizing the R352C mutant channel that exhibits hydrolysis-dependent open channel conductance: unequal single-channel current amplitudes between pre-and posthydrolytic states (24). Login to comment
153 Effects of Vx-770 on R352C-CFTR. Login to comment
154 (A and B) Representative single-channel traces for R352C-CFTR in the absence (A) or presence (B) of 200 nM Vx-770. Login to comment
157 Summary of the effects of Vx-770 on the gating patterns exhibited in R352C-CFTR and W401F/R352C-CFTR Total Experimental condition Without Vx-770* R352C (%) 834 (55) 301 (20) 173 (11) 39 (3) 169 (11) 1,516 (100) R352C/W401F (%) 733 (44) 326 (19) 122 (7) 28 (2) 474 (28) 1,683 (100) With 200 nM Vx-770 R352C (%) 578 (57) 126 (12) 73 (7) 20 (2) 217 (21) 1,014 (100) R352C/W401F (%) 411 (37) 162 (15) 79 (7) 31 (3) 425 (38) 1,108 (100) Five different categories of the gating pattern are illustrated. Login to comment
161 *Data for R352C-CFTR and R352C/W401F-CFTR in the absence of Vx-770 were taken from ref. 24. gate in the absence of ATP, C2 ࢒ O2) primarily affected by Vx-770 are supposed to take place in CFTR`s TMDs (24). Login to comment
180 As shown in Fig. S3 and Table 1, the W401F mutation almost doubles the reentry frequency of R352C-CFTR and also significantly enhances the effect of Vx-770 (Fig. 5, Table 1 and Fig. S3). Login to comment
207 Because the single-channel conductance is reduced by the R352C mutation, a pipette solution with 375 mM Cl-was used (in millimoles): Fig. 5. Login to comment
209 Representative single-channel traces for R352C/W401F-CFTR treated with 2.75 mM ATP in the absence (A) or presence (B) of 200 nM Vx-770. Login to comment
210 Frequency of opening bursts containing multiple rounds of O1 ࢐ O2 transition (summarized in Table 1) is increased and the overall open time is prolonged (Fig. S3D) compared with R352C-CFTR recorded in the same condition. Login to comment
213 For inside-out configuration, the 150 mM Cl- perfusion solution contained (in millimoles): 150 NMDG-Cl, 2 MgCl2, 10 EGTA, and 8 Tris (pH 7.4 with NMDG), the 375 mM Cl- perfusion solution used for R352C-CFTR contained (in millimoles): 375 NMDG-Cl, 2 MgCl2, 10 EGTA, and 8 Tris (pH 7.4 with NMDG). Login to comment
223 For kinetic analysis of R352C-CFTR, three current levels (C, O1, and O2) were determined from all point histograms. Login to comment

PMID: 23709221 [PubMed] Cui G et al: "Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function."

No. Sentence Comment

72 Open burst durations and closed durations were measured from single channel recordings of WT-, R352C-, D993C-, and R352C/D993C-CFTR, and then histograms and fits of them with single exponential functions were generated with IGOR (WaveMetrics, Inc., Lake Oswego, OR) to determine time constants for open burst durations (঄o, also called beta) and closed durations (঄c, also called ॷ) for all of the constructs. Login to comment
152 We generated R352C/D993C-CFTR and exposed the channels to MTS-2-MTS; MTS-2-MTS was chosen for this experiment because it leads to cross-linking of cysteines at a distance of b03;4.6 &#c5;, which is within the average distance for known salt bridges in a varietyofproteins(13).Priortostudieswiththedoublemutant,we also investigated each single mutant and their responses to monofunctional sulfhydryl-modifying reagents. Login to comment
162 Recovery of Charge at R352C and D993C Rescued Channel Stability in the Full Open State-R352C-CFTR exhibited single channel behavior similar to that previously reported for R352A-, R352Q-, and R352E-CFTR (13). Login to comment
163 A representative recording is shown in Fig. 5A, taken from one membrane patch bearing R352C-CFTR before and after exposure to MTSEAaf9; and after wash out. Login to comment
165 Prior to exposure to MTSEAaf9; , R352C-CFTR exhibited multiple conductance states, including closed (c) and s1, s2, and f open states. Login to comment
166 R352C-CFTR channels opened to all open states for very short durations (Fig. 5B). Login to comment
167 After exposure to MTSEAaf9; , R352C-CFTR channels exhibited mainly the f state with much longer mean burst duration and appearance of the s1 and s2 states as rare events, indicating recovery of open state stability (Fig. 5B). Login to comment
171 In contrast, deposition of negative charge at R352C-CFTR by exposure to MTSESafa; did not alter channel behavior (supplemental Fig. 2A). Login to comment
174 We note that in R352C-CFTR on the WT-CFTR background, exposure to MTSEAaf9; and MTSETaf9; led to an increase in NPo (Fig. 5A), reflecting modification of endogenous cysteines. Login to comment
176 In contrast to these results for R352C-CFTR, the stability of single channel opening in D993C-CFTR was rescued to mimic that of WT-CFTR by exposure to MTSESafa; (but not MTSEAaf9; or MTSETaf9; ), leading to significantly increased mean burst duration (supplemental Fig. 3B). Login to comment
179 We repeated the above experiments in R352C/Cys-less V510A-CFTR and D993C/ Cys-less V510A-CFTR to further rule out the possibility of any endogenous cysteines being involved in the process. Login to comment
182 A Bifunctional MTS Reagent Can Latch R352C/D993C-CFTR into the Full Open State Even after Washout of ATP-We hypothesized that the CFTR channel pore could be latched into the open state by cross-linking the two cysteines at R352C and D993C. Login to comment
183 We first tested the effects of monofunctional reagents MTSETaf9; , MTSEAaf9; , and MTSESafa; on the double mutant R352C/D993C-CFTR. Login to comment
184 None of these reagents rescued salt Dynamic Modulation of the CFTR Pore by Salt Bridges JULY 12, 2013ߦVOLUME 288ߦNUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20763 bridge function to stabilize channel behavior in R352C/D993C-CFTR in terms of stable openings to the f state (data not shown). Login to comment
185 Before applying MTS-2-MTS to the R352C/D993C-CFTR double mutant, we first tested the effects of this bifunctional linker on WT-CFTR (Fig. 6A) and Cys-less V510A-CFTR (Fig. 6B). Login to comment
187 We then examined the effects of MTS-2-MTS on R352C-D993C-CFTR (on the WT-CFTR background); a representative experiment is shown in Fig. 7. Login to comment
188 In the presence of ATP and PKA, prior to the addition of MTS-2-MTS, R352C/D993C-CFTR exhibited low open probability, unstable openings to the f state, and occasional subconductance open states. Login to comment
191 It seems likely that this reflects the fact that there are several possible consequences of exposing the double mutant to MTS-2-MTS, including covalent modification of R352C and D993C separately by two MTS-2-MTS molecules within each CFTR protein. Login to comment
195 Deposition of positive charge at R352C improved stability of the open state. Login to comment
196 A, sample traces from R352C-CFTR recorded from one patch under control conditions (top trace, ATP af9; PKA) and in the presence of 100 òe;M MTSEAaf9; (middle trace, ATP af9; PKA af9; MTSEAaf9; ) and then after washout with a large volume of intracellular solution and the subsequent addition of ATP and PKA alone (bottom trace, ATP af9; PKA) in excised inside-out membrane patches. Login to comment
198 All traces were recorded at VM afd; afa;100 mV. B, comparison of mean burst duration of R352C in the absence of MTSEAaf9; (ATP af9; PKA only) (afa;MTSEA) and in the presence of MTSEAaf9; with ATP af9; PKA (af9;MTSEA). Login to comment
209 The free energy change èc;èc;G between WT-CFTR and R352C/D993C-CFTR was afa;1.508 kcal/mol, which suggests that R352C and D993C interact with each other when CFTR is in the open state (supplemental Fig. 5). Login to comment
218 A, effects of 100 òe;M MTS-2-MTS on R352C-D993C-CFTR. Login to comment
220 B, MTS-2-MTS failed to functionally modify R352C/D993C-CFTR when applied when the channel was in the closed state. Login to comment

PMID: 23784545 [PubMed] Cai Z et al: "Acute inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel by thyroid hormones involves multiple mechanisms."

No. Sentence Comment

257 Recognizing that sojourns to subconductance states in Cysless-R352C-CFTR mimic the behavior of wild-type CFTR blocked by MOPS, Jih et al. (33) developed the energetic coupling model of CFTR channel gating (for review, see Ref. 32). Login to comment

PMID: 24727426 [PubMed] Wang Y et al: "Understanding how cystic fibrosis mutations disrupt CFTR function: from single molecules to animal models."

No. Sentence Comment

1915 However, Jih et al. (2012) interpreted the sojourns of the CFTR construct Cysless-R352C-CFTR to sub-conductance states to suggest that more than one ATP molecule might be hydrolysed during an open channel burst. Login to comment

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

No. Sentence Comment

268 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
321 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

PMID: 25512598 [PubMed] Yeh HI et al: "Modulation of CFTR gating by permeant ions."

No. Sentence Comment

272 Lately, by studying single-channel gating events of a CFTR mutant, R352C/Q, which exhibits unequivocal hydrolysis-dependent transitions of two distinct open states, Jih et al. (2012a) proposed an energetic coupling model featuring a more relaxed relationship between ATP hydrolysis in NBDs and opening/closing of the gate in CFTR`s TMDs. Login to comment

PMID: 26229102 [PubMed] Corradi V et al: "Cystic Fibrosis Transmembrane Conductance Regulator (CFTR): CLOSED AND OPEN STATE CHANNEL MODELS."

No. Sentence Comment

268 To rationalize the effects of small, positively charged reagents when a cysteine replaces Arg-352 (72), we can hypothesize that the shorter adduct (-CH2CH2-NH3 af9; ) on the cysteine might restore a functionally important positive charge in a position similar to that present in the native arginine, whereas the bulkier adduct (-CH2CH2-N(CH3)3 af9; ) might also sterically destabilize the McjD-like open conformation, given the relative narrowing of this region of the pore. Login to comment