ABCMdb

ABCC7 p.Leu102Cys

None has been submitted yet.

Publications

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

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

42 Interestingly, cysteines substituted into another pore-lining TM, TM1, did not show access to both sides of the membrane, with the outermost site in this TM that could be modified by intracellular MTS reagents (L102C) being insensitive to extracellular MTS reagents (18). Login to comment
43 In the present work, we have compared changes in the accessibility of T338C in TM6 with L102C in TM1 to both intracellular and extracellular cysteine-reactive reagents, both large, impermeant [2-sul- fonatoethyl] MTS (MTSES) and smaller, permeant Au(CN)2 - ions, under conditions in which ATP-dependent channel gating is altered. Login to comment
50 Two reporter cysteines in the pore were studied: T338C in TM6, which is modified by both intracellular and extracellular MTS reagents (17), and L102C in TM1, which is modified by intracellular, but not extracellular MTS reagents (18). Login to comment
52 These two reporter cysteine substitutions were combined with mutations in the NBDs that affect ATP-dependent channel gating: K464A (NBD1) and E1371Q (NBD2). Login to comment
79 Fig. 1 shows the influence of these NBD mutations on the rate of modification of two cysteines introduced deep into the channel pore from the inside, T338C in TM6 and L102C in TM1. Login to comment
82 Rate of modification of T338C and L102C by internal MTSES. Login to comment
86 The decline in current amplitude following MTSES application has been fitted by a single exponential function. C, average modification rate constants (k) for MTSES, calculated from fits to data such as those shown in A and B. Asterisks indicate a significant difference from the cysteine mutants T338C and L102C (black bars) (p Ͻ 0.05). Login to comment
89 For modification of T338C, the mean modification rate constant was decreased ϳ2.4-fold in a K464A background and increased ϳ3.9-fold in E1371Q, whereas the modification rate constant for L102C was decreased by ϳ26% in K464A and increased ϳ2.0-fold in E1371Q. Login to comment
90 As in our previous work (22), the effects of the E1371Q mutation were mimicked by locking channels open by treatment with 2 mM pyrophosphate (data not shown; ϳ3.9-fold increase for T338C and ϳ1.6-fold increase for L102C). Login to comment
92 To investigate whether permeant anions show the same regulated access from the cytoplasm to T338C and L102C, we investigated channel modification by Au(CN)2 - , a highly permeant anion that has been used previously to modify cysteine side chains in the CFTR pore (14, 22). Login to comment
93 As shown in Fig. 2, application of a low concentration of Au(CN)2 - (2 ␮M) caused a rapid inhibition of current carried by both T338C and L102C. Login to comment
94 As with MTSES, the rate of modification by Au(CN)2 - was significantly decreased by the K464A mutation (by ϳ1.8-fold for T338C and ϳ3.4-fold for L102C) and significantly increased by the E1371Q mutation (by ϳ5.6-fold for T338C and ϳ1.8-fold for L102C), as well as by pyrophosphate treatment (by ϳ6.0-fold for T338C and ϳ2.0-fold for L102C; data not shown). Login to comment
96 Regulated Access from Extracellular Solution to Pore- T338C is modified not only by intracellular, but also by extracellular MTS reagents (14, 17, 26), whereas L102C was reported to be insensitive to extracellular MTS reagents (18). Login to comment
98 Expression of all CFTR constructs (except those containing the E1371Q mutation, see below) in baby hamster kidney cells led to the appearance of cAMP-activated whole cell currents that were inhibited by the specific CFTR inhibitor GlyH-101 (Fig. 3 and supplemental Fig. S2) and which were not observed in cells transfected with vector alone (supplemental Fig. S2). Login to comment
99 Expression of all E1371Q-CFTR constructs led to the appearance of constitutive, cAMP-insensitive but GlyH-101-inhibited whole cell currents (supplemental Fig. S2). Login to comment
101 In contrast, T338C was strongly inhibited by very much lower concentrations of MTSES (1 ␮M) and Au(CN)2 - (200 nM) (Fig. 3B). Login to comment
102 L102C was insensitive to 2 mM MTSES, but was strongly inhibited by intermediate concentrations of Au(CN)2 - (10 ␮M) (Fig. 3C). Login to comment
109 The decline in current amplitude following Au(CN)2 - application has been fitted by a single exponential function. C, average modification rate constants(k)forAu(CN)2 - ,calculatedfromfitstodatasuchasthoseshowninAandB.AsterisksindicateasignificantdifferencefromthecysteinemutantsT338C and L102C (black bars) (p Ͻ 0.02). Login to comment
112 L102C was not apparently modified by extracellular MTSES (Fig. 3C), consistent with previous findings (18). Login to comment
113 Fig. 5 shows a similar analysis of the rate of modification by extracellular Au(CN)2 - , both for T338C (Fig. 5A; 200 nM Au(CN)2 - ) and for L102C (Fig. 5B; 10 ␮M Au(CN)2 - ). Login to comment
114 Quantification of the rate constant for modification (Fig. 5C) suggests, for modification of T338C, an increase of ϳ5.7-fold in K464A and a decrease of ϳ150-fold in E1371Q, and for modification of L102C, an increase of ϳ2.3-fold in K464A and a decrease of ϳ2.7-fold in E1371Q. Login to comment
116 Changing Patterns of Accessibility Suggest Marker Cysteine Residues "Switch Sides" of Membrane during Gating-The effects of NBD mutations on the rate of modification of T338C and L102C by internal cysteine-reactive reagents (estimated from experiments on inside-out membrane patches) and by external cysteine-reactive reagents (estimated from whole cell current recording experiments) are compared in Fig. 6. Login to comment
120 In each panel, it can be seen that the rate of modification by internal MTSES and Au(CN)2 - increases in the order K464A Ͻ Cys-less Ͻ E1371Q, whereas modification by extracellular MTSES (in T338C) and Au(CN)2 - shows the opposite pattern, K464A Ͼ Cys-less Ͼ E1371Q. Login to comment
122 This suggests that the reporter cysteines in the pore that we have used, T338C and L102C, are capable of "moving" from a relatively internally accessible position to a relatively externally accessible position. Login to comment
127 Sample whole cell currents were recorded at ϩ30 mV for Cys-less (A), T338C (B), and L102C (C). Login to comment
131 C, L102C currents were insensitive to MTSES (2 mM) but were inhibited by GlyH-101 (50 ␮M) and Au(CN)2 - (10 ␮M). Login to comment
151 For example, L102C in TM1 is modified by internal, but not external MTS reagents (18), a result confirmed by the present results (Figs. 1 and 3), whereas R104C, only 2 residues closer to the external end of TM1, is modified by external, but not internal MTS reagents (28). Login to comment
152 L102C, like T338C, becomes apparently more accessible to internal cysteine reactive reagents in open channels (Fig. 6B), but is inaccessible to extracellular MTSES (Fig. FIGURE 4. Login to comment
157 Modification rate constant for T338C/E1371Q was quantified from experiments using a higher concentration of MTSES (200 ␮M). Login to comment
158 Asterisks indicate a significant difference from T338C alone (p Ͻ 0.0005). Login to comment
160 L102C is accessible to permeant Au(CN)2 - ions applied to either side of the membrane, as expected for a permeant probe that ought to access the entire permeation pathway, and as with T338C access from the outside decreases as access from the inside increases (Fig. 6D), again consistent with easier access from the cytoplasm in open channels and from the extracellular solution in closed channels. Login to comment
161 One possible explanation for the difference in external accessibility of L102C in TM1 and T338C in TM6 is that T338C is located in a more superficial position in the outer mouth of the pore (at least in closed channels), such that it can be accessed by large extracellular MTS reagents that cannot penetrate further into the pore from the outside to modify L102C (Fig. 7A). Login to comment
162 Consistent with this differential access from the outside, the rate of modification by extracellular Au(CN)2 - is approximately 35 times greater for T338C than for L102C in a Cys-less background (Fig. 5C). Login to comment
170 Rate of modification of T338C and L102C by external Au(CN)2 - . Login to comment
174 Modification rate constants for T338C/E1371Q and L102C/E1371Q were quantified from experiments using a higher concentration of Au(CN)2 - (100 ␮M). Login to comment
175 Asterisks indicate a significant difference from T338C and L102C alone as applicable (black bars) (p Ͻ 0.01).Data are mean from three or four patches. Login to comment
178 Each panel illustrates the change in modification rate constant for the same reporter cysteine (T338C in A and C, L102C in B and D) in three different backgrounds (K464A, Cys-less, and E1371Q), for modification by MTSES (A and B) or Au(CN)2 - (C and D) applied to the intracellular (●, inside) or extracellular (E, outside) side of the membrane. Login to comment
180 Note that L102C was not apparently modified by extracellular MTSES (B; see Fig. 3C). Login to comment
191 In this model, reduced access from the extracellular solution in open channels is due to partial closure of a vestigial open gate, which decreases the rate of entry of extracellular MTSES and Au(CN)2 - to T338C and L102C, although not completely occluding the pore and thus allowing Cl- permeation. Login to comment
201 FIGURE7.ModelsofCFTRporestructureduringgating.Theimplicationsof the current experimental findings, that T338C and L102C in the CFTR pore show increased access to the extracellular solution in the closed state and increased access to the intracellular solution in the open state, can be interpreted according to a number of different simple diagram models of channel function. Login to comment
44 Interestingly, cysteines substituted into another pore-lining TM, TM1, did not show access to both sides of the membrane, with the outermost site in this TM that could be modified by intracellular MTS reagents (L102C) being insensitive to extracellular MTS reagents (18). Login to comment
45 In the present work, we have compared changes in the accessibility of T338C in TM6 with L102C in TM1 to both intracellular and extracellular cysteine-reactive reagents, both large, impermeant [2-sul- fonatoethyl] MTS (MTSES) and smaller, permeant Au(CN)2 afa; ions, under conditions in which ATP-dependent channel gating is altered. Login to comment
85 Rate of modification of T338C and L102C by internal MTSES. Login to comment
95 As in our previous work (22), the effects of the E1371Q mutation were mimicked by locking channels open by treatment with 2 mM pyrophosphate (data not shown; b03;3.9-fold increase for T338C and b03;1.6-fold increase for L102C). Login to comment
97 To investigate whether permeant anions show the same regulated access from the cytoplasm to T338C and L102C, we investigated channel modification by Au(CN)2 afa; , a highly permeant anion that has been used previously to modify cysteine side chains in the CFTR pore (14, 22). Login to comment
107 L102C was insensitive to 2 mM MTSES, but was strongly inhibited by intermediate concentrations of Au(CN)2 afa; (10 òe;M) (Fig. 3C). Login to comment
118 L102C was not apparently modified by extracellular MTSES (Fig. 3C), consistent with previous findings (18). Login to comment
119 Fig. 5 shows a similar analysis of the rate of modification by extracellular Au(CN)2 afa; , both for T338C (Fig. 5A; 200 nM Au(CN)2 afa; ) and for L102C (Fig. 5B; 10 òe;M Au(CN)2 afa; ). Login to comment
128 This suggests that the reporter cysteines in the pore that we have used, T338C and L102C, are capable of "moving" from a relatively internally accessible position to a relatively externally accessible position. Login to comment
133 Sample whole cell currents were recorded at af9;30 mV for Cys-less (A), T338C (B), and L102C (C). Login to comment
137 C, L102C currents were insensitive to MTSES (2 mM) but were inhibited by GlyH-101 (50 òe;M) and Au(CN)2 afa; (10 òe;M). Login to comment
167 L102C is accessible to permeant Au(CN)2 afa; ions applied to either side of the membrane, as expected for a permeant probe that ought to access the entire permeation pathway, and as with T338C access from the outside decreases as access from the inside increases (Fig. 6D), again consistent with easier access from the cytoplasm in open channels and from the extracellular solution in closed channels. Login to comment
168 One possible explanation for the difference in external accessibility of L102C in TM1 and T338C in TM6 is that T338C is located in a more superficial position in the outer mouth of the pore (at least in closed channels), such that it can be accessed by large extracellular MTS reagents that cannot penetrate further into the pore from the outside to modify L102C (Fig. 7A). Login to comment
169 Consistent with this differential access from the outside, the rate of modification by extracellular Au(CN)2 afa; is approximately 35 times greater for T338C than for L102C in a Cys-less background (Fig. 5C). Login to comment
177 Rate of modification of T338C and L102C by external Au(CN)2 d1a; . Login to comment
181 Modification rate constants for T338C/E1371Q and L102C/E1371Q were quantified from experiments using a higher concentration of Au(CN)2 afa; (100 òe;M). Login to comment
182 Asterisks indicate a significant difference from T338C and L102C alone as applicable (black bars) (p b0d; 0.01).Data are mean from three or four patches. Login to comment
185 Each panel illustrates the change in modification rate constant for the same reporter cysteine (T338C in A and C, L102C in B and D) in three different backgrounds (K464A, Cys-less, and E1371Q), for modification by MTSES (A and B) or Au(CN)2 afa; (C and D) applied to the intracellular (cf;, inside) or extracellular (E, outside) side of the membrane. Login to comment
187 Note that L102C was not apparently modified by extracellular MTSES (B; see Fig. 3C). Login to comment
198 In this model, reduced access from the extracellular solution in open channels is due to partial closure of a vestigial open gate, which decreases the rate of entry of extracellular MTSES and Au(CN)2 afa; to T338C and L102C, although not completely occluding the pore and thus allowing Clafa; permeation. Login to comment
208 FIGURE7.ModelsofCFTRporestructureduringgating.Theimplicationsof the current experimental findings, that T338C and L102C in the CFTR pore show increased access to the extracellular solution in the closed state and increased access to the intracellular solution in the open state, can be interpreted according to a number of different simple diagram models of channel function. 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

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
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
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
149 In TM6, I344 and V345 were proposed to lie on either side of this barrier, based on similar and L102C were all strongly inhibited by the application of the test dose of MTSES, suggesting that they were not covalently modified by MTSES pretreatment. 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
214 In open CFTR channels, internally applied MTS reagents can penetrate far enough into the pore as to modify L102C in TM1 and F337C in TM6. 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
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: 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
52 (B) Cytoplasmic application of MTSES dramatically reduced ATP-gated L102C/Cysless channel currents in an excised inside-out membrane patch. Login to comment
81 Fig. 1 B shows a representative recording of L102C mutant channels in response to the application of MTSES. 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
149 For instance, for L102C mutant channels, we found that macroscopic currents were increased by 108.5 5 9.5% (n &#bc; 6) after modification by MTSETapplied to the cytoplasmic side of the channel in excised inside-out patches (Fig. 5 A). Login to comment
150 Also similar to what has been reported for positions I344 and M348 in TM6, following MTSET modification of L102C-CFTR, robust activity was observed even in the complete absence of ATP. Login to comment
155 The remarkable effects of MTSET on L102C-CFTR currents shown in Fig. 5 A prompted us to examine this effect more closely with single-channel recordings. Login to comment
156 Fig. 5 B shows single-channel traces before and after MTSET modification of L102C-CFTR. Login to comment
159 Although previous studies have provided evidence that these short-lived closures are ATP independent (43,44) and could result from voltage-dependent block of the pore by large anions from the cytoplasmic side of the channel (45), it is noted that these events are abundantly present in the double mutant L102C/E1371Q at both negative and positive membrane potentials in the absence of ATP (see Fig. S3). Login to comment
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
174 Similar results were obtained from experiments on L102C (Fig. 6 B) and Q98C mutants. Login to comment
175 These data suggest that the modification rate of cysteines on these positions is FIGURE 5 Gating of MTSET-modified L102C/Cysless channel. Login to comment
176 (A) A continuous current recording of L102C/Cysless channels showing that MTSET modification increases the macroscopic current and renders the current ATP-independent. Login to comment
177 (B) Single-channel recording of the L102C/ Cysless-CFTR. 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
204 (B) A real-time recording with a similar protocol shown in (A) for L102C/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
274 Once we accept the possibility that TM1 undergoes major conformational changes during gating transitions, it is not surprising to see alterations in gating by the mutation itself (e.g., L102C), as well as by subsequent chemical modifications (Fig. 5). Login to comment
275 It is nevertheless interesting to note that in L102C/E1371Q-CFTR, the gate can open and close repeatedly even when the NBDs are in a dimeric configuration (Fig. S3). Login to comment
276 On the other hand, modification of L102C by MTSET literally renders the channel permanently open even long after ATP is removed and presumably NBDs have been separated. Login to comment