ABCMdb

ABCC7 p.Glu54Cys

None has been submitted yet.

Publications

PMID: 11468285 [PubMed] Fu J et al: "Cysteine substitutions reveal dual functions of the amino-terminal tail in cystic fibrosis transmembrane conductance regulator channel gating."

No. Sentence Comment

2 We introduced cysteines at two of these positions (E54C/D58C) and tested a series of methanethiosulfonate (MTS) reagents for their effects on the gating properties of these cysteine mutants in intact Xenopus oocytes and excised membrane patches. Login to comment
5 The inhibition of the opening rate of the E54C/D58C mutant channel by MMTS could be reversed by the reducing agent dithiothreitol (200 ␮M) or by elevating the bath ATP concentration above that required to activate maximally the wild type channel (>1 mM). Login to comment
43 EXPERIMENTAL PROCEDURES Mutagenesis-The E54C and E54C/D58C CFTR mutants were generated by PCR mutagenesis. Login to comment
87 The cRNAs encoding wild type CFTR, a single cysteine mutant (E54C), or a double cysteine mutant (E54C/D58C) were injected into oocytes and assayed by two-electrode voltage clamp analysis. Login to comment
90 As will be shown below, E54C/D58C also exhibits reduced channel Po and briefer channel openings in excised membrane patches relative to the wild type channel. Login to comment
95 The representative current-voltage relationship shown in Fig. 2C indicates that the MMTS-induced inhibition of the currents mediated by E54C/ D58C was due to lowered macroscopic conductance (i.e. reduced slope) rather than to a change in concentration driving force (i.e. altered reversal potential). Login to comment
98 Greater inhibition of the currents mediated by E54C/D58C was observed at higher MMTS concentration (1-5 mM), but nonspecific effects on wild type CFTR became significant at these considerably higher doses (results not shown). Login to comment
102 Covalent Modification of E54C/D58C in Excised Membrane Patches, Differential Responses to MMTS, MTSET, and MTSCE-Since the activity of the double cysteine mutant was substantially affected by thiol modification in intact oocytes, we performed a series of patch clamp studies of the E54C/D58C channel in excised inside-out patches. Login to comment
104 The unmodified E54C/D58C mutant channel exhibited an ϳ50% lower Po than WT CFTR (Fig. 3) under conditions that maximally activate the wild type channel (80 units/ml PKA; 1.0 mM MgATP). Login to comment
107 We next tested the effects of the neutral MMTS on the gating properties of E54C/D58C in excised patches, since this compound inhibited the macroscopic currents mediated by the cysteine mutants in intact oocytes. Login to comment
110 This effect on E54C/D58C channel activity in excised patches occurred within 1-5 min of adding MMTS to the patch (results not shown). Login to comment
113 If the effect of MMTS on the gating of the E54C/D58C channel is due to the formation of a mixed disulfide at these positions, then this effect should be reversed by a reducing agent such as DTT. Login to comment
114 Fig. 4 shows that the inhibitory effects of MMTS on the gating of E54C/D58C were completely reversed by the subsequent addition of 200 ␮M DTT. Login to comment
116 At this low concentration DTT had negligible effects on the gating of the wild type channel (results not shown) or on the gating of E54C/D58C in the absence of MMTS (Fig. 4, A and B). Login to comment
117 The fact that DTT alone had no effect on the gating of E54C/ FIG. 1. Login to comment
128 Although these charged reagents had no effect on the activity of the double cysteine mutant when applied extracellularly to intact oocytes (Fig. 2D), each inhibited E54C/D58C channel activity when applied to the cytoplasmic face of an inside-out patch. Login to comment
131 Thus, modification of the E54C/D58C channel with the positively charged MTS reagent not only inhibited channel opening rate but also further shortened the channel openings in excised membrane patches. Login to comment
138 More cRNA was injected for the mutants than for WT CFTR to achieve approximately the same absolute current levels following activation with the cAMP mixture (2 ng for E54C/D58C; 1 ng for E54C and E54A/D58A, and 0.5 ng for WT CFTR). Login to comment
139 C, representative I-V curves before and after MMTS (10 ␮M) modification of the E54C/D58C mutant channel. Login to comment
140 D, lack of effect of extracellular MTSET and MTSCE (100 ␮M each) on E54C/D58C CFTR currents. Login to comment
142 MMTS inhibits E54C/D58C channel activity in excised inside-out membrane patches primarily by decreasing channel opening rate. Login to comment
144 C, representative channel records showing the marked inhibition of E54C/ D58C channel activity in excised patches by 10 ␮M MMTS. Login to comment
148 E54C/D58C mutant channel. Login to comment
156 Fig. 8 shows that the activity (Po) of the E54C/D58C mutant channel can also be stimulated by the addition of 3 mM AMP-PNP (in the presence of 1 mM ATP) and that this effect is due to a large increase in open channel burst duration. This is qualitatively similar to the effect of AMP-PNP on wild type CFTR, although the degree of activation for the cysteine mutant was somewhat smaller than that previously observed for the wild type channel (wild type Po increases to nearly 0.9 under these conditions (14)). Login to comment
158 The positively charged MTSET inhibits the opening rate and burst duration of E54C/D58C channels in excised patches. Login to comment
159 A, representative records showing lack of effect of MTSET (100 ␮M) on WT CFTR channel activity. B, representative records showing marked inhibition of E54C/D58C channel activity by MTSET in excised membrane patch. Login to comment
161 DTT reverses the inhibitory effect of MMTS on E54C/D58C channel activity. Login to comment
162 A, representative channel records showing negligible effects of 200 ␮M DTT on the channel activity of unmodified E54C/D58C CFTR (no MMTS). Login to comment
164 Records were obtained before and 3-5 min after MMTS or DTT addition to the bath. C, mean data (Ϯ S.E.) showing the lack of effect of DTT on the Po, burst duration, and opening rate of the unmodified E54C/D58C channel. Login to comment
165 D, mean data (Ϯ S.E.) showing the recovery of the Po and opening rate of the MMTS-modified E54C/D58C channel by the subsequent addition of 200 ␮M DTT. Login to comment
167 cysteine mutant channel: (i) to determine if activating this channel with AMP-PNP protects against the inhibitory effects of MMTS modification on E54C/D58C channel activity and (ii) to determine if thiol modification of the N-tail cysteines influences subsequent activation by AMP-PNP. Login to comment
169 MMTS still markedly reduced the Po of the E54C/D58C channel, although in this case the inhibition by MMTS was due to reductions in both channel opening rate and burst duration. Login to comment
173 Conversely, AMP-PNP had a dramatic effect on the gating of the E54C/D58C channel when this cysteine mutant was first modified with the negatively charged MTSCE (Fig. 9). Login to comment
175 Such exceptionally long bursts are characteristic of the wild type channel when exposed to AMP-PNP under these conditions (14, 22) but are never observed for the unmodified E54C/D58C channel or the corresponding alanine mutants (14). Login to comment
178 The rationale for these experiments were 2-fold: (i) CFTR channel opening is activated by ATP binding to one or both NBDS (5-8), and (ii) each MTS reagent inhibited the channel opening rate of the double cysteine mutant. Fig. 10 shows that increasing the ATP concentration to 10 mM following modification of the E54C/D58C channel with MMTS nearly completely reversed the inhibitory effects of thiol modification on Po and channel opening rate. Login to comment
184 The negatively charged MTSCE inhibits opening rate but induces the appearance of long open channel bursts for E54C/D58C CFTR. Login to comment
185 A, representative records showing the prolonged (Ͼ10 s) open channel bursts that occur following modification of E54C/ D58C channels in excised patches by 100 ␮M MTSCE. Login to comment
187 Histograms of burst durations recorded for the unmodified E54C/D58C channel and following modification of this mutant with MTSET or MTSCE. Login to comment
196 Stimulation of the E54C/D58C channel with AMP-PNP does not protect against subsequent inhibition by MMTS. Login to comment
200 B, mean data showing the effects of AMP-PNP followed by MMTS addition on the Po, burst duration, and opening rate of E54C/D58C CFTR. Login to comment
202 Thiol modification of the E54C/D58C channel alters the subsequent response to AMP-PNP. Login to comment
203 A, representative records showing sequential effects of MMTS (10 ␮M) and AMP-PNP (3 mM) on E54C/D58C channel activity. B, mean data showing effects of MMTS followed by AMP-PNP on Po, burst duration and opening rate for the cysteine mutant. Login to comment
204 C, representative records showing the exceptionally long bursts that are induced by AMP-PNP when the E54C/D58C channel has been modified with the negatively charged MTSCE. Login to comment
207 The opening rate of the E54C/D58C channel can be rescued from MMTS inhibition by increasing the bath Mg-ATP concentration. Login to comment
208 A, representative records showing the effect of elevating the bath Mg-ATP concentration on the unmodified E54C/D58C channel. Login to comment
213 This inhibition could also be reversed by DTT at a concentration (200 ␮M) below that which affects the activity of the unmodified E54C/D58C channel (Fig. 4) or the wild type channel. Login to comment
224 The inhibition of opening rate by thiol modification of the E54C/D58C mutant channel could be reversed by elevating the bath ATP concentration beyond that normally required to maximally activate the wild type channel. Login to comment
233 Although the major effect of MMTS on E54C/D58C gating was to decrease channel opening rate, this reagent also inhibited the response of the double cysteine mutant to the poorly hydrolyzable AMP-PNP. Login to comment
234 AMP-PNP stimulates the activity of wild type CFTR and, to a lesser extent, the E54C/D58C mutant by stabilizing channel openings (i.e. by increasing burst duration). Login to comment
236 In contrast to their similar effects on channel opening rate, the three MTS reagents had qualitatively different effects on the duration of channel openings exhibited by the E54C/D58C mutant. Login to comment
238 Covalent modification with the negatively charged MTSCE also recovered the very long open channel bursts (Ͼ1 min) that are normally exhibited by the wild type channel (but not unmodified E54C/D58C) following exposure to AMP-PNP. Login to comment