ABCMdb

PMID: 11468285

Fu J, Kirk KL
Cysteine substitutions reveal dual functions of the amino-terminal tail in cystic fibrosis transmembrane conductance regulator channel gating.
J Biol Chem. 2001 Sep 21;276(38):35660-8. Epub 2001 Jul 23., 2001-09-21 [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 4 ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala The MTS reagents had negligible effects on the gating of the wild type channel or a corresponding double alanine mutant (E54A/D58A) under the same conditions. Login to comment 5 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 16 ABCC7 p.Asp58Asn A disease-associated mutant that maps to one of these sites (D58N CFTR) exhibited similar alterations in macroscopic current kinetics and open channel burst duration (14). Login to comment 32 ABCC7 p.Asp58Asn Although we failed to observe a dramatic effect of mutations in this region on channel opening rate, we did observe a modest increase in interburst duration (i.e. closed time) for D58N CFTR (14) as if this mutation also affected the ability of the channel to open. Login to comment 40 ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala This inhibition of opening rate was observed at concentrations of MTS reagents that had no effect on the gating of the wild type channel or of a corresponding alanine mutant (E54A/D58A). Login to comment 43 ABCC7 p.Glu54Cys ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys EXPERIMENTAL PROCEDURES Mutagenesis-The E54C and E54C/D58C CFTR mutants were generated by PCR mutagenesis. Login to comment 87 ABCC7 p.Glu54Cys ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 89 ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala In addition, like the corresponding double alanine mutant (E54A/D58A) (13), the double cysteine mutant deactivated faster than wild type CFTR fol- lowing removal of the cAMP-activating mixture (Fig. 1C). Login to comment 90 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 96 ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala We also tested the effects of MMTS on the wild type channel (WT CFTR) and the double alanine mutant (E54A/D58A) to determine if the inhibition was specifically due to modification of the engineered cysteines at residues 54 and 58. Login to comment 97 ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala At this concentration MMTS had small effects (5-10% inhibition) on the macroscopic currents mediated by WT CFTR and E54A/ D58A in intact oocytes (Fig. 2, A and B) and had no effects on the channel activities of these constructs in excised patches (see below). Login to comment 98 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 108 ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala When added to the cytoplasmic face of excised patches, MMTS had little effect on the channel open probability and gating kinetics (opening rate, burst duration) of either wild type CFTR or the double alanine mutant (E54A/D58A) (Fig. 3, A and B). Login to comment 110 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys The fact that DTT alone had no effect on the gating of E54C/ FIG. 1. Login to comment 126 ABCC7 p.Asp58Cys 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. Login to comment 128 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys C, representative I-V curves before and after MMTS (10 ␮M) modification of the E54C/D58C mutant channel. Login to comment 140 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys D, lack of effect of extracellular MTSET and MTSCE (100 ␮M each) on E54C/D58C CFTR currents. Login to comment 142 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys MMTS inhibits E54C/D58C channel activity in excised inside-out membrane patches primarily by decreasing channel opening rate. Login to comment 143 ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala A and B, representative channel records showing negligible effects of 10 ␮M MMTS on the gating of WT CFTR and E54A/D58A CFTR. Login to comment 144 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys C, representative channel records showing the marked inhibition of E54C/ D58C channel activity in excised patches by 10 ␮M MMTS. Login to comment 148 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys E54C/D58C mutant channel. Login to comment 156 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys The positively charged MTSET inhibits the opening rate and burst duration of E54C/D58C channels in excised patches. Login to comment 159 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys DTT reverses the inhibitory effect of MMTS on E54C/D58C channel activity. Login to comment 162 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 182 ABCC7 p.Asp58Asn We also observed that a disease-associated mutant (D58N) that maps to this region exhibited unstable openings (i.e. shortened open channel burst duration) in excised patches (14). Login to comment 184 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys The negatively charged MTSCE inhibits opening rate but induces the appearance of long open channel bursts for E54C/D58C CFTR. Login to comment 185 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys Stimulation of the E54C/D58C channel with AMP-PNP does not protect against subsequent inhibition by MMTS. Login to comment 200 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys Thiol modification of the E54C/D58C channel alters the subsequent response to AMP-PNP. Login to comment 203 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys A, representative records showing the effect of elevating the bath Mg-ATP concentration on the unmodified E54C/D58C channel. Login to comment 212 ABCC7 p.Asp58Ala ABCC7 p.Glu54Ala This inhibitory effect was not observed for the wild type channel or for a corresponding double alanine mutant (E54A/D58A), which argues against a nonspecific effect of these reagents on channel activity. Login to comment 213 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 ABCC7 p.Glu54Cys ABCC7 p.Asp58Cys 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 239 ABCC7 p.Asp58Asn These data are consistent with the shorter channel openings that were previously observed for the disease-associated D58N mutant (14), which harbors a structurally subtle substitution with the exception of the loss of negative charge at this position (asparagine for aspartate). Login to comment