ABCMdb

ABCC7 p.Ile344Cys

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Publications

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

100 The oocytes 750 500 250 0 µS 180012006000 s IBMX MTSEA Cd 2+ DTT 200 100 0 µS 180012006000 s IBMX DTT Cd 2+ MTSEA A B C -100 -80 -60 -40 -20 0 20 40 % Change in conductance Y325C A326C L327C I328C K329C G330C I331C I332C L333C R334C K335C I336C F337C T338C T339C I340C S341C F342C WT I344C V345C R347C M348C A349C V350C T351C Q353C * * * * * Cd 2+ 1mM MTSEA 1mM D FIGURE 1. Login to 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: 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
281 Note the lack of consistent results reported for F337C, S341C, I344C, R347C, T351C, R352C, and Q353C (shaded). 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

17 For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. Login to comment
107 Spontaneous ATP-independent gating of cysless/I344C and cysless/M348C was also increased by MTSET because after the removal of ATP, there remained a substantial amount of current, which can be inhibited by CFTR-specific inhibitor, K335C, F337, and T338C at 50 mV membrane potential (0.46 pA for cysless/WT). Login to comment
179 (B) The Po, obtained from the traces in A, of the cysless/I344C channel before (black) and after (blue) modification. Login to comment
181 n = 7 for cysless/ I344C. Login to comment
183 (C) Single-channel amplitude of the cysless/I344C channel before and after modification. Login to comment
186 Instead, we will focus on the four other positive hits (i.e., I344C, V345C, M348C, and Q353C). Login to comment
208 In a representative recording of a patch containing hundreds of cysless/I344C channels (Fig. 10), one can clearly discern the single-channel amplitude from the expanded trace before and after phosphorylation-dependent activation. Login to comment
241 The modification rate by 1 mM MTSES when Figure 10. MTSES decreased single-channel amplitude of cysless/I344C channels. Login to comment
274 (B) A continuous single-channel recording of cysless/I344C showing a dramatic increase of the spontaneous ATP-independent gating after MTSET modification. Login to comment
314 However, our results showing drastic alterations in gating kinetics caused by modulating TM6 in the TMDs (e.g., a long-lasting opening on the order of tens of seconds with the MTSET-modified cysless/I344C channel) raise the possibility that gating motion in the TMDs can also affect ATP binding and hydrolysis in the NBDs (compare Kogan et al. 2001), a subject worth more extensive future explorations. Login to comment
316 In fact, our data show that the Po of spontaneous gating in the absence of ATP (Bompadre et al., 2007; Wang et al., 2010) is visibly increased by MTSET modification (Figs. 4 D and 12 B; Po is 0.19 ± 0.04, n = 5 for cysless/M348C, and 0.63 ± 0.03, n = 4 for cysless/ I344C). Login to comment
317 The single-channel trace of cysless/I344C in Fig. 12 B illustrates a negligible ATP-independent gating before MTSET modification, whereas frequent opening and closing events in the absence of ATP can Locher, 2006) as a template. Login to comment

PMID: 21796338 [PubMed] Qian F et al: "Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant."

No. Sentence Comment

140 In this respect, the slow rate of modification observed in N1138C (Fig. 3b) is similar to that we reported for P99C and L102C in TM1 [41] and T338C and S341C in TM6 [9], and the much higher modification rate constant for T1142C, S1141C, and (to a lesser extent) M1140C is closer to that reported for K95C in TM1 [41] and I344C, V345C, and M348C in TM6 [9]. Login to comment
207 However, charge-conservative mutations in the analgous part of TM6-for example, in I344C, V345C, M348C, and A349C-also failed to significantly alter Cl-conductance [4]. Login to comment

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

No. Sentence Comment

481 One construct, I344C, exhibited an increase in was elegantly demonstrated by Horn and co-workers (163) who showed that cysteine residues engineered intoconductance when exposed to MTSEA0 . Login to comment

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

No. Sentence Comment

164 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
166 We introduced the R352Q mutation into the Cysless/ I344C-CFTR channel. 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
174 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 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 (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
165 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
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
175 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
178 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
180 (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

PMID: 22303012 [PubMed] Wang W et al: "Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7)."

No. Sentence Comment

148 Residues closer to the cytoplasmic end of TM6 show regulated access to intracellular MTS reagents without being accessible to extracellular MTS reagents, for example I344C is accessible to intracellular MTSES only in open channels (22) but is not modified by extracellular MTS reagents (14, 17). Login to comment
154 Residues closer to the cytoplasmic end of TM6 show regulated access to intracellular MTS reagents without being accessible to extracellular MTS reagents, for example I344C is accessible to intracellular MTSES only in open channels (22) but is not modified by extracellular MTS reagents (14, 17). Login to comment

PMID: 22234285 [PubMed] Wang W et al: "Conformational change opening the CFTR chloride channel pore coupled to ATP-dependent gating."

No. Sentence Comment

3 The rate of modification of Q98C (TM1) and I344C (TM6) by both [2-sulfonatoethyl] methanethiosulfonate (MTSES) and permeant Au(CN)2 - ions was reduced when ATP concentration was reduced from 1 mM to 10 μM, and modification by MTSES was accelerated when 2 mM pyrophosphate was applied to prevent channel closure. Login to comment
6 The rate of modification of Q98C and I344C by both MTSES and Au(CN)2 - was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected. Login to comment
52 Example timecourses of macroscopic currents (measured at -50 mV during brief voltage excursions from a holding potential of 0 mV) carried by K95C, Q98C, I344C and V345C as indicated, in inside-out membrane patches. Current amplitudes were measured every 6 s following attainment of stable current amplitude after channel activation. Channels were activated with PKA (20 nM) and either a high concentration of ATP (1 mM; in (A) and (C)-(E)) or a low concentration of ATP (10 μM; (B)). Login to comment
55 In each panel, MTSES (20 μM for K95C, I344C and V345C, and 200 μM for Q98C; see Materials and methods) was applied to the cytoplasmic face of the patch at time zero (as indicated by the hatched bar at the bottom of each panel). Login to comment
60 Additional mutations were introduced into the cys-less background using the QuikChange site-directed mutagenesis -200 -150 -100 -50 0 I (pA) Time (s) K95C A) 1 mM ATP -180 -120 -60 0 Q98C -400 -300 -200 -100 0 -200 -150 -100 -50 0 I344C -300 -200 -100 0 -500 -400 -300 -200 -100 0 V345C -250 -200 -150 -100 -50 0 -300 -200 -100 0 -600 -400 -200 0 -750 -500 -250 0 -600 -400 -200 0 -800 -600 -400 -200 0 I (pA) Time (s) 20 µM MTSES 20 µM MTSES 20 µM MTSES200 µM MTSES C) 1 mM ATP + 2 mM PPi E) E1371Q (1 mM ATP) I (pA) Time (s) D) K464A (1 mM ATP) B) 10 µM ATP -100 -75 -50 -25 0 -200 -150 -100 -50 0 -80 -60 -40 -20 0 -80 -60 -40 -20 0 -120 -90 -60 -30 0 -80 -60 -40 -20 0 -60 -40 -20 0 -300 -200 -100 0 I (pA) Time (s) I (pA) Time (s) 0 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 system (Agilent Technologies, Santa Clara, CA, USA) and verified by DNA sequencing. Login to comment
91 As described previously, at K95C and Q98C (TM1) and at I344C and V345C (TM6), current amplitude is decreased by treatment 100 1000 10000 1 mM ATP 10 µM ATP 1 mM ATP + 2 mM PPi K95C Q98C I344C V345C * * ModificationRateConstant(M-1 s-1 )ModificationRateConstant(M-1 s-1 ) A B K95C Q98C I344C V345C 100 1000 10000 Cys-less +K464A +E1371Q * * * * * * Fig. 3. Login to comment
100 Inspection of these example timecourses indicates that, while such manipulations have no effect on the rate of modification in K95C or V345C, the rate of modification is altered in both Q98C and I344C (Fig. 2A-C). Login to comment
101 Quantification of the mean modification rate constant (as described in Materials and methods) demonstrates that decreasing ATP concentration from 1 mM (Fig. 2A) to 10 μM (Fig. 2B) to decrease channel opening rate significantly decreases the rate of modification in Q98C and I344C (~2.0-fold decrease in modification rate constant; Pb0.01), whereas the rate of modification of K95C and V345C was apparently unaffected (P>0.2) (Fig. 3A). Login to comment
102 Conversely, treatment with PPi (2 mM; Fig. 2C) to inhibit channel closure and increase open probability significantly increases the rate of modification in Q98C and I344C (2.5-2.8-fold increase in modification rate constant; Pb0.01) but has no effect on the rate of modification of K95C and V345C (P>0.4) (Fig. 3A). Login to comment
103 These results suggest that pharmacological manipulation of NBD function results in changes in the accessibility of Q98C and I344C-but not K95C or V345C-to cytoplasmic MTSES. Login to comment
111 The K464A mutation significantly decreased the rate of MTSES modification at Q98C and I344C (2.5-2.9-fold decrease in modification rate constant; Pb0.005) but had no effect on the rate of modification at K95C or V345C (P>0.5) (Fig. 3B). Login to comment
112 Conversely, the E1371Q mutation significantly increased the rate of MTSES modification at Q98C and I344C (3.0-3.1-fold increase in modification rate constant; Pb0.02) but had no effect on the rate of modification at K95C or V345C (P>0.25) (Fig. 3B). Login to comment
113 These results therefore suggest that altering NBD function non-pharmacologically by mutagenesis alters accessibility of Q98C and I344C to cytoplasmic MTSES, whereas accessibility of K95C and V345C are unaffected by NBD-driven channel gating. Login to comment
145 In fact, we found that currents carried by K95C, Q98C, and I344C were potently inhibited by much lower concentrations of Au(CN)2 - (200 nM-2 μM; Fig. 6). Login to comment
146 Both K95C and I344C were rapidly inhibited by 200 nM Au(CN)2 - (Fig. 6), reflecting a high modification rate constant (Fig. 7). Login to comment
150 We therefore compared the rate of Au(CN)2 - inhibition in K95C, Q98C and I344C at two different ATP concentrations (10 μM and 1 mM), as well as in channels also bearing the NBD mutations K464A or E1371Q (Fig. 6). Login to comment
151 Quantification of the mean modification rate constant demonstrated that decreasing ATP -300 -200 -100 0 -400 -300 -200 -100 0 -200 -150 -100 -50 0 -60 -40 -20 0 -120 -80 -40 0 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 0 60 120 180 -100 -75 -50 -25 0 -90 -60 -30 0 -90 -60 -30 0 -120 -80 -40 0 A) 1 mM ATP I (pA) I (pA) I (pA) Time (s) 200 nM Au(CN)2 C) K464A (1 mM ATP) D) E1371Q (1 mM ATP) 2 µM Au(CN)2 200 nM Au(CN)2 I (pA) Time (s) Time (s) -200 -150 -100 -50 0 -90 -60 -30 0 -120 -80 -40 0 K95C Q98C I344C B) 10 µM ATP Time (s) Fig. 6. Timecourse of modification by Au(CN)2 - . Login to comment
153 Reporter cysteines (K95C, Q98C, and I344C as indicated) were examined in isolation (A, B) or combined with the NBD mutations K464A (C) or E1371Q (D). Login to comment
154 In each panel, Au(CN)2 - (200 nM for K95C and I344C, and 2 μM for Q98C; see Materials and methods) was applied to the cytoplasmic face of the patch at time zero (as indicated by the hatched bar at the bottom of each panel). Login to comment
156 concentration to 10 μM significantly decreased the rate of Au(CN)2 - modification of Q98C and I344C (1.7-1.8-fold decrease in modification rate constant; Pb0.005) but had no effect on the rate of modification at K95C (P>0.4) (Fig. 7). Login to comment
157 In addition, the K464A mutation significantly decreased the rate of Au(CN)2 - modification of Q98C and I344C (1.4-1.5-fold decrease in modification rate constant with 1 mM ATP; Pb0.005) but had no effect on the rate of modification at K95C (P>0.5) (Fig. 7). Login to comment
158 Conversely, the E1371Q mutation significantly increased the rate of Au(CN)2 - modification at Q98C and I344C (2.8-3.5-fold increase in modification rate constant; Pb0.01) but had no effect on the rate of modification at K95C (P>0.2) (Fig. 7). Login to comment
166 In contrast, nearby residues (Q98C in TM1, I344C in TM6) showed strongly state-dependent accessibility, both to the large MTSES (Fig. 3) and the small, permeant Au(CN)2 - ion (Fig. 7). Login to comment
168 Given the well known effects of these manipulations on channel gating and overall open probability, it seems reasonable to suggest that the rate of modification at Q98C and I344C is positively associated with open probability, suggesting that open channels are modified more easily than are closed channels. Login to comment
174 However, our measurements of time constants of changes in macroscopic current amplitude in channels that are opening and closing do not allow us to estimate the rate of modification at Q98C and I344C in closed channels. Login to comment
175 Previously we suggested that the rate of modification of Q98C [15] and I344C [14] is negligible in inactive channels prior to PKA-dependent phosphorylation. Login to comment
176 It is possible that phosphorylation leads to partial opening of the putative gate and a partial increase in access, and ATP-dependent gating then results in a further increase in access through this region. This could allow slow modification of Q98C and I344C in phosphorylated, but closed, channels. Login to comment
181 Perhaps surprisingly, then, we find the effects of low ATP and the K464A mutation on modification rate constants for Q98C and I344C to be quantitatively similar and in the range of 1.5 to 3-fold (Figs. 3, 7). Login to comment
201 While K95C, Q98C and I344C were rapidly inhibited by low concentrations of cytoplasmic Au(CN)2 - (Fig. 6), V345C showed similar Au(CN)2 - sensitivity as cys-less CFTR (data not shown). Login to comment

PMID: 22042986 [PubMed] Bai Y et al: "Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7)."

No. Sentence Comment

198 (C) Second-order rate constants (MTSES  ) of Texas red MTSEA+ modification for cysless/ S1141C-, cysless/N1148C-, cysless/ I344C-, and cysless/M348C-CFTR channels. Login to comment

PMID: 21746847 [PubMed] Wang W et al: "Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

22 Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. Login to comment
96 Fig. S2 shows the lack of sensitivity to the reducing agent DTT of macroscopic currents carried by the double-cysteine mutant channels K95C/ I344C and Q98C/I344C. Login to comment
139 Our work concerning intracellular MTS reagent modification in TM6 also identified some cysteines that could be modified in both activated and nonactivated channels (e.g., V345C and M348C), and others that could apparently be modified only after channel activation (e.g., T338C, S341C, and I344C), suggesting a state-dependent conformational change that alters access of internally applied MTS reagents into the pore (El Hiani and Linsdell, 2010). Login to comment
162 (A-C) Example leak-subtracted I-V relationships for K95C/I344C (A), Q98C/I344C (B), and Q98C/M348C (C) after channel activation with 20 nM PKA and 1 mM ATP. Login to comment
166 Note that cys-less CFTR, the single mutants K95C, Q98C, or I344C, and the double mutant Q98C/M348C were all insensitive to CuPhe under these conditions. Login to comment
167 Also note that CuPhe had a stronger inhibitory effect on currents carried by K95C/I344C when measured at +80 mV compared with 80 mV; this same apparent voltage dependence was previously reported for K95C/S1141C under similar experimental conditions (Zhou et al., 2010). Login to comment
168 In contrast, the inhibitory effects of CuPhe on Q98C/I344C were similar when measured at 80 mV or +80 mV. Login to comment
170 (E) Mean effects of CuPhe (black bars), CuPhe followed by washing with normal bath solution (white bars), and CuPhe followed by DTT (gray bars) on macroscopic current amplitude in K95C/I344C (left) and Q98C/I344C (right) at +80 mV. Login to comment
172 Mean of data from three to seven patches is shown in D and E. both K95C/I344C and Q98C/I344C by CuPhe was not reversed by washing CuPhe from the bath; however, partial reversal was seen when 5 mM DTT was applied in the continued presence of CuPhe (Fig. 6, A, B, and E), consistent with CuPhe inhibition of these channels reflecting some oxidative process. Login to comment
173 The results shown in Fig. 6 suggest that disulfide bond formation can occur between K95C and I344C and between Q98C and I344C after channel activation. Login to comment
175 After patch excision, inside-out patches from cells expressing either K95C/I344C or Q98C/ I344C were treated with cytoplasmic CuPhe for 2 min, after which CuPhe was washed from the bath and currents were activated using PKA and ATP, as usual. Login to comment
178 Both K95C/I344C and Q98C/I344C channel currents were also potently inhibited by the addition of Cu2+ ions alone (without phenanthroline) to the bath (Fig. 8). Login to comment
180 Each of the single mutants K95C, Q98C, and I344C showed reversible "paired" mutants with one cysteine introduced into each of TM1 (at K95 or Q98) and TM6 (at I344 and V345). Login to comment
182 However, K95C/ I344C, Q98C/I344C, and Q98C/M348C did generate macroscopic PKA- and ATP-dependent currents in inside-out patches. Login to comment
184 However, the oxidizing agent CuPhe, which has previously been used to induce disulfide bond formation between introduced cysteines in other parts of the CFTR protein (Mense et al., 2006; Loo et al., 2008; Serohijos et al., 2008; Zhou et al., 2010), led to a strong reduction in current amplitude in both K95C/I344C and Q98C/I344C (Fig. 6). Login to comment
185 Neither cys-less CFTR nor the single mutants K95C, Q98C, or I344C appeared sensitive to CuPhe under these conditions (Fig. 6 D), consistent with this agent acting by causing disulfide bond formation between the two introduced cysteine side chains in the double mutants K95C/I344C and Q98C/I344C. Login to comment
188 (A and B) Example leak-subtracted I-V relationships for K95C/I344C (A) and Q98C/I344C (B) after channel activation with 20 nM PKA and 1 mM ATP in inside-out patches that had been pretreated with CuPhe for 2 min, and then washed to remove CuPhe. Login to comment
200 Our results concerning the accessibility of cysteines introduced into TM1 are summarized, and compared inhibition by Cu2+ that was of intermediate potency between the cys-less background and the double mutants K95C/I344C and Q98C/I344C. Login to comment
206 (A) Example leak-subtracted I-V relationships for cys-less (left), I344C (center), and Q98C/I344C (right) after channel activation with 20 nM PKA and 1 mM ATP. Login to comment
208 In all channel constructs studied, these inhibitory effects of Cu2+ were readily and rapidly reversed by washing Cu2+ from the bath (for example, see right panel for complete reversal of the strong blocking effect on Q98C/I344C). Login to comment
209 (B) Mean fractional current remaining after the addition of different concentrations of Cu2+ for cys-less (), I344C (), and Q98C/I344C (). Login to comment
210 Data are fitted as described in Materials and methods, giving Kd = 129 µM and nH = 1.36 for cys-less, Kd = 19.5 µM and nH = 1.21 for I344C, and Kd = 3.91 µM and nH = 1.65 for Q98C/I344C. Login to comment
242 Changes in channel function after the addition of the oxidizing agent CuPhe, that were not reversed by removal of this agent, were taken as evidence for the formation of a disulfide bridge between K95C in TM1 and I344C in TM6, and between Q98C in TM1 and I344C in TM6 (Fig. 6). Login to comment
256 For comparison, the MTSES modification rate constant for P99C and L102C (Fig. 3) was similar to that of T338C and S341C in TM6 (El Hiani and Linsdell, 2010) (all between 100 and 150 M1 s1 ), and the modification rate constant for K95C was comparable to, or slightly greater than, that of I344C, V345C, and M348C (El Hiani and Linsdell, 2010) (all between 2,000 and 4,000 M1 s1 ). 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

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

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

114 Another mutant, I344C, reacted with MTSEA+ more slowly, requiring an 8-min application of 2.5 mM MTSEA+ to significantly alter the current (Fig. 5 B). Login to comment
135 The slow rate of reaction of MTSEA' with the I344C mutant and the inability of the larger, anionic MTSES- to react with 1344C suggests that access of the reagents to this residue may be sterically limited, possibly by neighboring residues on other membrane-spanning segments. Login to comment
113 Another mutant, I344C, reacted with MTSEA+ more slowly, requiring an 8-min application of 2.5 mM MTSEA+ to significantly alter the current (Fig. 5 B). Login to comment
133 The slow rate of reaction of MTSEA' with the I344C mutant and the inability of the larger, anionic MTSES- to react with 1344C suggests that access of the reagents to this residue may be sterically limited, possibly by neighboring residues on other membrane-spanning segments. Login to comment

PMID: 23083715 [PubMed] El Hiani Y et al: "Tuning of CFTR chloride channel function by location of positive charges within the pore."

No. Sentence Comment

126 This relative location of amino acids is also supported by experimental evidence that disulfide bonds can be formed between cysteine side chains substituted for K95 and S1141 (8), as well as between K95C and I344C, and between Q98C and I344C (13). Login to comment

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

No. Sentence Comment

185 Likewise in Bai et al. (8), chemical modifications of an engineered cysteine (I344C or M348C) in TM6 drastically increase ATP-independent activity to the level of ATP-dependent activity before modifications. Login to comment

PMID: 23442957 [PubMed] Gao X et al: "Cysteine scanning of CFTR's first transmembrane segment reveals its plausible roles in gating and permeation."

No. Sentence Comment

164 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

PMID: 25143385 [PubMed] El Hiani Y et al: "Metal bridges illuminate transmembrane domain movements during gating of the cystic fibrosis transmembrane conductance regulator chloride channel."

No. Sentence Comment

51 To investigate potential Cd2af9; bridges formed between pore-lining cysteine side chains exposed in the inner vestibule of the CFTR pore, we combined individual cysteines that we previously found to be accessible to cytoplasmically applied methanethiosulfonate reagents in three important pore-lining TMs: TM1 (K95C, Q98C) (13), TM6 (I344C, V345C, M348C, A349C) (15), and TM12 (M1140C, S1141C, T1142C, Q1144C, W1145C, V1147C, N1148C) (16), to generate a total of 50 double cysteine mutants (8 TM1:TM6; 14 TM1:TM12; 28 TM6:TM12). Login to comment
71 In contrast, the remaining seven double cysteine mutants, namely I344C/S1141C (Fig. 2, C and D), V345C/S1141C, M348C/ S1141C (Fig. 2, C and E), M348C/V1144C, M348C/W1145C, M348C/V1147C, and M348C/N1148C, all showed increased sensitivity to Cd2af9; , leading to a significant decrease in Ki as compared with either of the single cysteine mutants from which they were derived (estimated Ki values b0d; 50 òe;M; Fig. 3). Login to comment
80 In each case, PPi treatment resulted in a weakening of Cd2af9; inhibition (Fig. 4A) and a significant increase in Ki (Fig. 4B) of between 2.3-fold (in I344C/S1141C) and 97-fold (in M348C/ S1141C). Login to comment
83 As shown in Fig. 5, all E1371Q-containing channels tested were only weakly sensitive to inhibition by Cd2af9; , resulting in a significant increase in Ki both in single cysteine (I344C, M348C, S1141C) and in double cysteine (I344C/S1141C, Fig. 5, A-C; M348C/S1141C, Fig. 5, A, D, and E) mutant channels. Login to comment
84 However, the effect of the E1371Q mutation was greater in the double cysteine mutants; this gating mutation increased Ki 30-fold in I344C/S1141C (Fig. 5C) and 2500-fold in M348C/S1141C (Fig. 5E). Login to comment
98 C, sample time courses (upper panels) and I-V curves (lower panels) recorded from similar experiments for the double cysteine mutants I344C/S1141C (left) and M348C/S1141C (right). Login to comment
137 Thus, M348C is able to form Cd2af9; bridges with cysteines at multiple positions in TM12 (S1141C, Q1144C, W1145C, V1147C, N1148C) (Fig. 8B), and S1141C is able to form Cd2af9; bridges with cysteines both in TM1 (K95C) and in TM6 (I344C, V345C, M348C) (Fig. 8C). Login to comment
164 A, sample time courses and I-V curves illustrating the low Cd2af9; sensitivity of constitutively active I344C/S1141C/E1371Q (left panels) and M348C/S1141C/E1371Q (right panels) channels in inside-out patches. Experiments were performed as described in the legend for Fig. 2. Login to comment

PMID: 25675504 [PubMed] Gao X et al: "Localizing a gate in CFTR."

No. Sentence Comment

68 (C) Reaction between I344C-CFTR and 1 mM [Au(CN)2]- in the presence or absence of ATP (see Results for details). Login to comment
70 (D) State-independent reactivity of I344C-CFTR to [Au(CN)2]- . Login to comment
192 [Au(CN)2]- , forskolin, with G1349D, /M/s Outside R334C 189 &#b1; 39 - 403 &#b1; 20 537 &#b1; 56 K335C - - 56 &#b1; 9 1,809 &#b1; 201 F337C 437 &#b1; 49 - 20 &#b1; 3 32 &#b1; 6 T338C 752 &#b1; 59 - 1,135 &#b1; 166 118 &#b1; 18 Inside I344C 32 &#b1; 5 37 &#b1; 4 - - N1148C 437 &#b1; 66 2,089 &#b1; 130 - - Residues located extracellularly (extra.) Login to comment