ABCC7 p.Asp58Cys
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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."
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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.
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ABCC7 p.Asp58Cys 11468285:2:56
status: NEW5 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).
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ABCC7 p.Asp58Cys 11468285:5:47
status: NEW43 EXPERIMENTAL PROCEDURES Mutagenesis-The E54C and E54C/D58C CFTR mutants were generated by PCR mutagenesis.
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ABCC7 p.Asp58Cys 11468285:43:54
status: NEW87 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.
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ABCC7 p.Asp58Cys 11468285:87:102
status: NEW90 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.
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ABCC7 p.Asp58Cys 11468285:90:29
status: NEW95 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).
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ABCC7 p.Asp58Cys 11468285:95:142
status: NEW98 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).
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ABCC7 p.Asp58Cys 11468285:98:52
status: NEW102 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.
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ABCC7 p.Asp58Cys 11468285:102:30
status: NEWX
ABCC7 p.Asp58Cys 11468285:102:287
status: NEW104 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).
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ABCC7 p.Asp58Cys 11468285:104:20
status: NEW107 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.
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ABCC7 p.Asp58Cys 11468285:107:80
status: NEW110 This effect on E54C/D58C channel activity in excised patches occurred within 1-5 min of adding MMTS to the patch (results not shown).
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ABCC7 p.Asp58Cys 11468285:110:20
status: NEW113 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.
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ABCC7 p.Asp58Cys 11468285:113:48
status: NEW114 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.
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ABCC7 p.Asp58Cys 11468285:114:71
status: NEW116 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).
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ABCC7 p.Asp58Cys 11468285:116:137
status: NEW126 D58C indicates that the lower channel activity of this mutant is unlikely due to formation of an intramolecular disulfide bond between the two engineered cysteines in the N-tail.
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ABCC7 p.Asp58Cys 11468285:126:0
status: NEW128 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.
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ABCC7 p.Asp58Cys 11468285:128:170
status: NEW131 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.
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ABCC7 p.Asp58Cys 11468285:131:31
status: NEW138 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).
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ABCC7 p.Asp58Cys 11468285:138:172
status: NEW139 C, representative I-V curves before and after MMTS (10 M) modification of the E54C/D58C mutant channel.
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ABCC7 p.Asp58Cys 11468285:139:91
status: NEW140 D, lack of effect of extracellular MTSET and MTSCE (100 M each) on E54C/D58C CFTR currents.
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ABCC7 p.Asp58Cys 11468285:140:80
status: NEW142 MMTS inhibits E54C/D58C channel activity in excised inside-out membrane patches primarily by decreasing channel opening rate.
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ABCC7 p.Asp58Cys 11468285:142:19
status: NEW144 C, representative channel records showing the marked inhibition of E54C/ D58C channel activity in excised patches by 10 M MMTS.
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ABCC7 p.Asp58Cys 11468285:144:73
status: NEW148 E54C/D58C mutant channel.
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ABCC7 p.Asp58Cys 11468285:148:5
status: NEW156 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)).
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ABCC7 p.Asp58Cys 11468285:156:48
status: NEW158 The positively charged MTSET inhibits the opening rate and burst duration of E54C/D58C channels in excised patches.
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ABCC7 p.Asp58Cys 11468285:158:82
status: NEW159 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.
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ABCC7 p.Asp58Cys 11468285:159:163
status: NEW161 DTT reverses the inhibitory effect of MMTS on E54C/D58C channel activity.
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ABCC7 p.Asp58Cys 11468285:161:51
status: NEW162 A, representative channel records showing negligible effects of 200 M DTT on the channel activity of unmodified E54C/D58C CFTR (no MMTS).
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ABCC7 p.Asp58Cys 11468285:162:125
status: NEW164 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.
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ABCC7 p.Asp58Cys 11468285:164:210
status: NEW165 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.
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ABCC7 p.Asp58Cys 11468285:165:102
status: NEW167 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.
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ABCC7 p.Asp58Cys 11468285:167:151
status: NEW169 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.
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ABCC7 p.Asp58Cys 11468285:169:47
status: NEW173 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).
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ABCC7 p.Asp58Cys 11468285:173:68
status: NEW175 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).
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ABCC7 p.Asp58Cys 11468285:175:178
status: NEW178 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.
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ABCC7 p.Asp58Cys 11468285:178:317
status: NEW184 The negatively charged MTSCE inhibits opening rate but induces the appearance of long open channel bursts for E54C/D58C CFTR.
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ABCC7 p.Asp58Cys 11468285:184:115
status: NEW185 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.
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ABCC7 p.Asp58Cys 11468285:185:125
status: NEW187 Histograms of burst durations recorded for the unmodified E54C/D58C channel and following modification of this mutant with MTSET or MTSCE.
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ABCC7 p.Asp58Cys 11468285:187:63
status: NEW196 Stimulation of the E54C/D58C channel with AMP-PNP does not protect against subsequent inhibition by MMTS.
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ABCC7 p.Asp58Cys 11468285:196:24
status: NEW200 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.
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ABCC7 p.Asp58Cys 11468285:200:122
status: NEW202 Thiol modification of the E54C/D58C channel alters the subsequent response to AMP-PNP.
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ABCC7 p.Asp58Cys 11468285:202:31
status: NEW203 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.
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ABCC7 p.Asp58Cys 11468285:203:103
status: NEW204 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.
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ABCC7 p.Asp58Cys 11468285:204:106
status: NEW207 The opening rate of the E54C/D58C channel can be rescued from MMTS inhibition by increasing the bath Mg-ATP concentration.
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ABCC7 p.Asp58Cys 11468285:207:29
status: NEW208 A, representative records showing the effect of elevating the bath Mg-ATP concentration on the unmodified E54C/D58C channel.
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ABCC7 p.Asp58Cys 11468285:208:111
status: NEW213 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.
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ABCC7 p.Asp58Cys 11468285:213:142
status: NEW224 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.
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ABCC7 p.Asp58Cys 11468285:224:65
status: NEW233 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.
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ABCC7 p.Asp58Cys 11468285:233:42
status: NEW234 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).
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ABCC7 p.Asp58Cys 11468285:234:84
status: NEW236 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.
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ABCC7 p.Asp58Cys 11468285:236:179
status: NEW238 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.
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ABCC7 p.Asp58Cys 11468285:238:198
status: NEW