ABCMdb

ABCC7 p.Lys95Cys

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Publications

PMID: 15634668 [PubMed] Linsdell P et al: "Location of a common inhibitor binding site in the cytoplasmic vestibule of the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

91 These results, using a number of different amino acid substitutions of Lys-95, strongly suggest that side chain charge at this position is important in controlling the apparent affinity of glibenclamide block; the apparent Kd at -100 mV was not affected in the charge-conservative K95R but was significantly increased in charge-neutralizing mutants (K95A, K95C, K95Q) and most strongly increased in the charge-reversing K95E mutant. Login to comment

PMID: 20142516 [PubMed] Zhou JJ et al: "Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore."

No. Sentence Comment

17 Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. Login to comment
131 (B) Mean fractional current remaining after the addition of CuPhe as a function of voltage in wild type (), K95C (), S1141C (), and K95C/S1141C (). Login to comment
132 Data values for K95C/S1141C were significantly different from wild type, K95C, or S1141C (P < 0.05 in each case) at all voltages examined. Login to comment
133 (C) Mean fractional current remaining after the addition of CuPhe at +80 mV for different channel variants as indicated, and for K95C/S1141C after washing with normal bath solution (wash) or with bath solution supplemented with 5 mM DTT (wash + DTT). Login to comment
135 (D) Example leak-subtracted macroscopic I-V relationships for cys-less K95C/S1141C-CFTR recorded under the same conditions as in A. Login to comment
144 In contrast, each of the mutants, K95C, S341C, and S1141C (all in a cys-less background), was strongly sensitive to both MTSES and MTSET. Login to comment
146 In contrast, MTSET inhibited currents carried by cys-less S341C and cys-less S1141C, but potentiated currents carried by cys-less K95C. Login to comment
148 In contrast, in K95C, deposition of positive charge by reaction with MTSET may replace the function of the positive charge at this site that is lost as a consequence of the K95C mutation. Login to comment
149 Consistent with this idea, MTSET modification converts the cys-less K95C I-V relationship from outwardly rectified (before modification) to linear or mildly inwardly rectified after modification (Fig. 4 B). Login to comment
152 The strong reactivity of cys-less K95C, S341C, and S1141C to intracellular MTSES and MTSET is consistent with the cysteine side chains introduced at these positions being exposed within the aqueous inner vestibule of the pore. Login to comment
154 The K95C/S1141C double mutant generated small macroscopic currents that showed outward rectification under symmetrical Cl concentration conditions (Fig. 3), as observed with all mutations that remove the charge at K95 (Linsdell, 2005), including K95C (Fig. 3). Login to comment
155 K95C/S1141C currents in inside-out patches were insensitive to the application of 5 mM of the reducing agent dithiothreitol (DTT; not depicted), suggesting that spontaneous disulfide bond formation between the two cysteine side chains is either negligible or without functional consequence. Login to comment
156 However, the oxidizing reagent CuPhe, which has 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), led to a strong reduction in current amplitude in K95C/ S1141C (Fig. 3, A-C), suggesting a functional modification of the protein. Login to comment
158 Interestingly, neither wild-type CFTR currents nor the single mutants K95C or S1141C appeared sensitive to CuPhe under these conditions (Fig. 3, A-C), consistent with this agent acting by causing cross-linking of the two cysteine side chains introduced at these positions. Login to comment
159 Inhibition of K95C/S1141C by CuPhe was only partially reversed by washing; however, the degree of reversibility was significantly enhanced by the inclusion of 5 mM DTT in the wash solution (Fig. 3 C), consistent with CuPhe inhibition reflecting some oxidative process. Login to comment
161 A K95C/S341C double mutant did not yield functional currents in inside-out patches either without or after treatment with 5 mM DTT. Login to comment
162 Although these results suggest that K95C can be cross-linked to S1141C, they are potentially confounded by the presence of endogenous cysteine side chains in the CFTR protein. Login to comment
164 As shown in Fig. 3 D, cys-less K95C/S1141C also generated small, outwardly rectified currents in inside-out membrane patches that showed the same apparent sensitivity to CuPhe as that described above for these mutations in a wild-type background. Login to comment
165 On average, the application of CuPhe reduced current amplitude in cys-less K95C/S1141C by 84.7 ± 5.2% at +80 mV (n = 5). Login to comment
167 K95C/S1141C channel investigated the S1141K mutant at the macroscopic current level using depolarizing voltage ramp protocols like those used in Fig. 1, it became apparent that channel function had been altered in a way we had not anticipated from our initial single-channel experiments (see Fig. 2). Login to comment
260 Furthermore, we suggest that irreversible inhibition of channel function in the K95C/S1141C double mutant by the oxidizing agent CuPhe (Fig. 3) most likely reflects formation of a disulfide bridge between these two pore-lining cysteine side chains (Fig. 4). 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
151 Thus, a disulfide bridge can be formed between K95C in TM1 and S1141C in TM12, suggesting that the β carbon distance is in the range of ~5-8 Å for these two introduced cysteines [45]. Login to comment

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

No. Sentence Comment

112 On mutants K95C and K335C interact with methanethiosulfo- the basis of these data, the minimum diameter of the nate (MTS) reagents, and mutations that eliminate the CFTR pore was estimated to be Ç5.3 A˚ (77), similar to positive charge at K335 reduce single-channel conduc- that reported for other Cl0 channels (10, 20, 55). Login to comment

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

No. Sentence Comment

475 In three TM1 mutants, G91C, K95C, Q98C, all of which fall on the same face of a predicted TM1 a- ter`` is close to the cytoplasmic end of the pore and that R352 may play a role in determining charge selectivity forhelix, the conductance was irreversibly altered by either MTSES0 or MTSEA0 . 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

130 Beck et al., (2008) also studied R334C CFTR channels expressed in Xenopus oocytes using a protocol similar to that employed here, but failed to detect increased reactivity toward externally-applied MTSEA+ in the activated state. Login to comment
158 Figure 3, C and D, contains the time courses for the reactions of [Au(CN)2]afa; with the F337C and T338C CFTRs before TABLE 1 EC50 at 0 mV (mean afe; S.E.M.) for GlyH-101 for wt and mutant CFTRs, with and without modification with iodoacetamide CFTR EC50 at 0 mV òe;M wt 1.1 afe; 0.11 (n afd; 4) K95C 1.4 afe; 0.35 (n afd; 4) F337C 1.8 afe; 0.06 (n afd; 3) F337C af9; iodoacetamide 2.4 afe; 0.29 (n afd; 3) T338C 3.7 afe; 0.27 (n afd; 3) T338C af9; iodoacetamide 24 afe; 2.6 (n afd; 3) Fig. 3. 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

4 Modification of K95C (TM1) and V345C (TM6) was not affected by these manoeuvres. 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
165 For two introduced cysteine residues-K95C in TM1 and V345C in TM6-the rate of modification by cytoplasmic reagents was independent of ATP-dependent channel gating (Figs. 3, 6), suggesting that access to these residues is similar both in open channels and in closed channels. Login to comment
173 As pointed out above, the lack of apparent state-dependence of modification in K95C and V345C suggests that the rate of modification at these sites is similar in closed channels. 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: 21746847 [PubMed] Wang W et al: "Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore."

No. Sentence Comment

19 Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Login to 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
71 In contrast, macroscopic currents carried by four mutants, K95C, Q98C, P99C, and L102C, were found to be significantly and rapidly sensitive to the application of both MTSES and MTSET (Figs. 1-3). 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
98 As shown in Fig. 3 A, MTSES modification was rapid in K95C, even when a low concentration of MTSES (20 µM) was used, and considerably slower in L102C (using 200 µM MTSES). Login to comment
105 (B) Example leak-subtracted I-V relationships for cys-less CFTR, K95C, Q98C, P99C, L102C, and R104C, recorded from inside-out membrane patches after maximal channel activation with 20 nM PKA, 1 mM ATP, and 2 mM PPi. Login to comment
112 As shown in Fig. 4 A, patches excised from MTSET-pretreated cells expressing K95C, Q98C, P99C, or L102C all gave macroscopic currents that were increased in amplitude after the addition of 2 mM constants was that modification was faster for cysteines introduced closer to the intracellular end of TM1, and slower for cysteines located more deeply along the axis of TM1 (Fig. 3 B). Login to comment
123 (A) Example time courses of macroscopic currents (measured at 50 mV during brief voltage excursions from a holding potential of 0 mV) carried by K95C (left) and L102C (right) as indicated, in inside-out membrane patches. Login to comment
125 In each case, MTSES (20 µM for K95C and 200 µM for L102C) 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
128 Asterisks indicate a significant difference from MTSES modification of K95C (P < 0.005), and daggers indicate a significant difference from MTSES modification of the same mutant (P < 0.05). Login to comment
136 Whereas K95C channels were again rendered insensitive to a test exposure to MTSES, again consistent with them having been covalently modified during pretreatment, currents carried by Q98C, P99C, MTSET to the intracellular solution. Login to comment
138 These results suggest that none of K95C, Q98C, P99C, or L102C can be modified covalently by extracellular MTSET. Login to comment
141 We used a similar approach to determine if K95C, Q98C, P99C, and L102C could be modified by MTSES pretreatment. Login to comment
148 Proximity and alignment of TMs 1 and 6 The results described in Fig. 5, suggesting that K95C is accessible to cytoplasmic MTSES in nonactivated channels but that Q98C is accessible only in activated channels, imply that K95 and Q98 may lie close to the putative barrier within the pore that we recently proposed to regulate access from the cytoplasmic solution (El Hiani and Linsdell, 2010). Login to comment
150 These results, which are summarized quantitatively in Fig. 5 C, suggest that although K95C can be modified by MTSES before channel activation, Q98C, P99C, and L102C are modified by MTSES only very slowly, if at all, in channels that have not been activated by PKA and ATP. 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
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
181 Unfortunately, the double mutants K95C/V345C and Q98C/V345C did not yield functional currents when expressed in BHK cells, even after treatment with DTT to break any possible disulfide bonds; a similar lack of functional expression was previously reported for K95C/S341C (Zhou et al., 2010). 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
228 Thus, the side chains of TM1 mutants K95C, Q98C, P99C, and L102C that we identified as accessible to MTS reagents applied from the inside (Fig. 2) were not accessible to MTSET applied to the outside (Fig. 4), whereas R104C, previously shown to be modified by external MTS reagents (Zhou et al., 2008), was not modified by internal MTSES or MTSET (Fig. 2). Login to comment
238 Although we have not investigated the state dependence of MTSES modification in TM1 in such great detail, our present results suggest a similar arrangement in which K95C can readily be modified before channel activation (Fig. 5), whereas Q98C, P99C, and L102C are modified rapidly after channel activation (Fig. 3) but very slowly if at all before activation (Fig. 5). 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
261 If this is the case, the similar discrepancy between MTSES and MTSET modification rate constants at all sites tested implies that the location of anion-cation discrimination that underlies this discrepancy may lie between the cytoplasmic mouth of the pore and the most accessible cysteine residue, namely, that at K95C. Login to comment
264 This kind of information is necessary to develop and validate three-dimensional structuralmodelsoftheporeregion.Previously,weshowed that a disulfide bond could be formed between K95C (in TM1) and S1141C (in TM12) (Zhou et al., 2010). Login to comment

PMID: 7515047 [PubMed] Akabas MH et al: "Amino acid residues lining the chloride channel of the cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

69 Application of the MTS reagents irreversibly alteredthe CFTR-induced currents of three of the cysteine substitution mutants, G91C, K95C, and Q98C. Login to comment
72 20 and 3C) and Q98C by 32 2 4% (n= 9) and potentiated the current of the mutant K95C by 108 2 22%(n = 4) (Figs. Login to comment
75 The anionic reagent, MTSES-, had no effect on the K95C and G91C mutants (Fig. 3,A andB).To determine whether thelack of effect was due to inability to react with Cys-95 or lack of effect followingreaction, we sequentially applied MTSES- and MTSEA+;MTSES- did not prevent the potentiationof the current by MTSEA+(data notshown). Login to comment
82 The effect ofthe MTS reagentson wild typeCFTR and on thethreechannel-liningmutants GSlC, K95C,and QSSC. Login to comment
86 A and C are from oocytesinjectedwith wild type CFTR B, Q98C; D,K95C; E, G91C. Login to comment
97 The application ofMTSEA' to the mutant K95C increased the CFTR-induced current. Login to comment
101 1 MINMTS-EA, 8 MIN C K95C 5 D K95C 5 I -50 0 50 100 150-50 0 50 100 150 CHANGEINCURRENT (X) FIG.3. Login to comment
110 Based on the accessibility of the cysteine-substitution mutants G91C,K95C and Q98C to the MTS reagents, we infer that the side chains of the corresponding wild type residues, Gly-91,Lys-95, and Gln-98, line the channel ofCFTR. 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: 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

11 Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR`s gating cycle. Login to comment
82 This observed decrease of macroscopic currents is due to covalent modification of the engineered cysteines by the reagent as the effect persisted even after a complete removal of MTSES. Similar observations were made for K95C- and Q98C-CFTR. Login to comment
108 We next tested the accessibility to external MTSES on three positions identified by experiments with inside-out patches, namely K95C, Q98C, and L102C, in the same manner and all three positions turned out nonreactive (data not shown). Login to comment
152 For Q98C and K95C mutant channels, the increases in the mean current amplitude following MTSET modification were ~2- and 6-fold, respectively. Login to comment
153 Because the single-channel conductance was drastically decreased in K95C-CFTR, we were not able to assess the gating effect of MTSET modification. Login to comment
165 State-dependent modification of E92C-, K95C-, Q98C-, and L102C-CFTR The observation that MTSET modification of Q98C and L102C alters CFTR gating suggests that TM1 indeed participates in gating motions of CFTR. Login to comment
172 However, Fig. 6 A shows a representative recording of current response for K95C-CFTR mutants. Login to comment
196 Third, in the report by Wang et al. (35), K95C, but not Q98C, P99C, or L102C, can react with internal MTSES even before the channel is activated by PKA and ATP, implying a regulated barrier between positions 95 and 98. Login to comment
203 (A) A representative recording for MTSES modification in the absence of ATP for K95C/Cysless channels. Login to comment
206 (C) Summary of the modification rates for K95C, Q98C, and L102C in the presence of ATP (solid squares). 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