ABCMdb

ABCC7 p.Ser1141Cys

None has been submitted yet.

Publications

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
139 In an interesting contrast to these effects, K95C/S1141C currents were not significantly affected by the addition of 300 µM Cd2+ ions (not depicted). 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
147 In S341C and S1141C, modification by the bulky MTSET molecule may partly occlude the pore, reducing Cl current. 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
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

5 Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation. Login to comment
80 Application of MTSES (200 μM) or MTSET (2 mM) to the intracellular solution after channel activation with PKA, ATP, and PPi significantly altered macroscopic current amplitude in nine out of 19 cysteine-substituted mutants tested (N1138C, M1140C, S1141C, T1142C, Q1144C, W1145C, V1147C, N1148C, and S1149C; Figs. 1 and 2). Login to comment
82 In the central region of TM12 (N1138C, M1140C, and S1141C), macroscopic current amplitude was decreased by both MTSES and MTSET (Figs. 1 and 2). Login to comment
92 For MTS reagent-sensitive TM12 mutants located relatively deeply into the pore from its cytoplasmic end (N1138C, M1140C, S1141C, and T1142C), the rate of modification was estimated from the time course of macroscopic current amplitude change following application of MTSES (20-200 μM). Login to comment
93 As shown in Fig. 3a, modification was rapid in M1140C, S1141C, and T1142C, even using a low concentration of MTSES (20 μM), and noticeably slower in N1138C, even with 200 μM MTSES. Login to comment
96 To gain some information on the orientation of TM12 in the CFTR pore, we therefore examined whether the outermost cysteines introduced into this TM that were sensitive to internal MTS reagents (N1138C, M1140C, and S1141C) could also be modified by extracellular MTSET. Login to comment
101 As shown in Fig. 4a, patches excised from MTSET-pretreated cells expressing N1138C, M1140C, or S1141C all gave macroscopic currents that were decreased in amplitude following addition of 2 mM MTSET to the intracellular solution. Login to comment
103 These results suggest that none of N1138C, M1140C, or S1141C can be modified covalently by extracellular MTSET. Login to comment
106 We used a similar approach to determine whether N1138C, M1140C, S1141C, and T1142C, located relatively deeply into the pore from its cytoplasmic end and all strongly sensitive to inhibition by intracellular MTSES (Figs. 2 and 3), could be modified by MTSES pretreatment. Login to comment
118 Both S1141C and T1142C channels were again rendered insensitive to the test exposure to MTSES, again consistent with them having been covalently modified during pretreatment. Login to comment
120 These results, which are summarized quantitatively in Fig. 5d, suggest that while S1141C and T1142C can be modified by MTSES prior to channel activation, N1138C and M1140C are modified by MTSES only very slowly, if at all, in channels that have not been activated by PKA and ATP. Login to comment
125 Extracellular MTSET did not appear able to modify the outermost of these internal MTS-sensitive cysteines (N1138C, M1140C, and S1141C; Fig. 4), consistent with MTS reagents not being permeant. Login to 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
143 a Example timecourses of macroscopic current amplitudes (measured at -50 mV) carried by N1138C, M1140C, S1141C, and T1142C as indicated in inside-out membrane patches. Login to comment
148 Using a similar approach, we find that in TM12, S1141C and T1142C can be readily modified by cytoplasmic MTSES prior to channel activation (Fig. 5), whereas N1138C and M1140C are modified rapidly after channel activation (Fig. 3) but very slowly, if at all, prior to channel activation (Fig. 5). 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: 22042986 [PubMed] Bai Y et al: "Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7)."

No. Sentence Comment

114 (A) Current recorded at 50 mV in inside-out membrane patches containing thousands of cysless/S1141C-CFTR channels. Login to comment
133 Consistent with this idea, the S1141C-CFTR channel, when modified by 2-aminocarbonylethyl MTS (MTSACE), a similarly sized but neutral reagent, also displayed a smaller single-channel current amplitude (Fig. 3 D). Login to comment
137 (A and B) Measurements of the second-order reaction rate constant (MTSES  ) of MTSES modification in the presence of 2 mM ATP for cysless/Q1144C(A)andcysless/S1141C (B). Login to comment
156 (C and D) Single-channel recordings show that the unitary current amplitude decreases after MTSET+ (C; blue) or MTSACE (D; green) exposure for cysless/S1141C. Login to comment
157 Linear fits to single-channel current measurements at different voltages yield unitary conductance values for cysless/S1141C of 7.0 pS before (C and D; black) and 6.1 pS after both MTSET+ (C; blue) and MTSACE (D; green) treatment. Login to comment
180 Note that the modification is faster in the presence of ATP than in the absence of ATP for cysless/N1148C-CFTR channels (B), whereas it is slower in the presence of ATP than in the absence of ATP for cysless/S1141C-CFTR channels (C). Login to comment
194 (A) Chemical modification of cysless/S1141C-CFTR channels by Texas red MTSEA+ (orange arrows) without ATP. Login to 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

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
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: 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
77 In particular, M348C/S1141C was associated with a dramatic increase in sensitivity to Cd2af9; (Fig. 2, C and E, and Fig. 3) with an estimated Ki of 0.14 afe; 0.02 òe;M (n afd; 5). 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
114 However, one mutant, K95C/S1141C, was apparently stimulated by Cd2af9; (Fig. 6A), and this stimulation was of a greater magnitude than that seen in the K95C single cysteine mutant (Figs. 6C and 7). Login to comment
115 This suggests that a Cd2af9; bridge may form between K95C in TM1 and S1141C in TM12. Login to comment
116 As with K95C itself, the stimulatory effects of Cd2af9; on K95C/S1141C were abolished by the E1371Q mutation (Fig. 6C) or by treatment with PPi (data not shown), suggesting that the observed effects of Cd2af9; are due to an increase in channel open probability and a stabilization of the channel open state. Login to comment
127 Note that the Ki for M348C/S1141C (0.14 afe; 0.02 òe;M, n afd; 5) is too small to be visible on this scale, but was significantly different from either M348C or S1141C alone (p b0d; 0.0005; see also Fig. 2E). 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
140 Similarly, S1141C is able to form Cd2af9; bridges with different sites in TM6 (Fig. 8C). Login to comment
146 Most striking in this respect was the M348C/S1141C mutant FIGURE 4. Login to comment
148 A, mean fractional current remaining following the addition of different concentrations of Cd2af9; in M348C/S1141C channels in the presence of PKA and ATP (F) or following activation by PKA and ATP followed by treatment with 2 mM PPi to maximize channel open probability (E). Login to comment
152 The unusually high apparent Cd2af9; binding affinity of this double cysteine mutant (Figs. 3B and 4B), more than 700-fold lower Ki than the corresponding single cysteine mutants (Figs. 2E and 3), suggests that M348C and S1141C are uniquely well positioned to coordinate tight Cd2af9; binding in closed channels. Login to comment
153 However, when the channel is open, these two cysteines do not appear to coordinate Cd2af9; at all because the apparent Cd2af9; affinity in M348C/S1141C/ E1371Q channels appears the same as in M348C/E1371Q or S1141C/E1371Q (Fig. 5, D and E). Login to comment
154 In contrast, a single identified Cd2af9; bridge between TMs 1 and 12 (K95C/S1141C) appears to stabilize the channel open state, leading to a significant increase in channel current (Fig. 6). Login to comment
157 If we assume that the effects of Cd2af9; bridges on the stability of the channel open and closed states reflects changes in the relative positions of cysteine side chains introduced into different TMs in these states, then our results would suggest that a number of side chains in TMs 6 and 12 are in close proximity in closed channels, and K95C (TM1) and S1141C (TM12) are in close proximity in open channels. 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
175 As described above, several different Cd2af9; bridges can form between M348C (TM6) and TM12 (Fig. 8, A and B), as well as between S1141C (TM12) and TM6; all appear to stabilize the closed state (Figs. 3 and 4). Login to comment
176 Thus, for the reasons described earlier, there may be FIGURE 6. Cadmium increases macroscopic current amplitude in K95C and K95C/S1141C channels in a gating-dependent manner. Login to comment
177 A, sample time courses and I-V curves showing Cd2af9; concentration-dependent increases in macroscopic current amplitude in K95C (left panels) and K95C/S1141C (right panels) channels in inside-out patches. Experiments were performed as described in the legend for Fig. 2. Login to comment
181 C, Cd2af9; causes a significantly greater increase in K95C/S1141C current amplitude as compared with K95C (p b0d; 0.0001); this increase is not observed in K95C/S1141C/E1371Q channels. Login to comment
188 In contrast to this consistent pattern of Cd2af9; bridge formation between TMs 6 and 12 stabilizing the closed state, a single Cd2af9; bridge between K95C (TM1) and S1141C (TM12) appears to stabilize the open state (Fig. 6). Login to comment
198 E, proposed graphic model for Cd2af9; bridge formation between the key sites illustrated in D in open and closed states, and proposed implications for conformationalrearrangementofthesethreeTMsassociatedwithchannelgating.TheclosedstateisstabilizedbyaCd2af9; bridgebetweenM348C(TM6)andS1141C (TM12), and the open state is stabilized by a Cd2af9; bridge between K95C (TM1) and S1141C (TM12). Login to comment