ABCMdb

PMID: 11867445

Harrington MA, Kopito RR
Cysteine residues in the nucleotide binding domains regulate the conductance state of CFTR channels.
Biophys J. 2002 Mar;82(3):1278-92., [PubMed]

Sentences

No. Mutations Sentence Comment

72 ABCC7 p.Cys524Ser ABCC7 p.Cys491Ser C491S, C524S, C1344/1355S, and C491/524S mutants were inserted into a pcDNA3.1 expression vector and transiently transfected into HEK 293 cells using calcium phosphate. Login to comment 73 ABCC7 p.Cys524Ser ABCC7 p.Cys491Ser C491S, C524S, C1344/1355S, and C491/524S mutants were inserted into a pcDNA3.1 expression vector and transiently transfected into HEK 293 cells using calcium phosphate. Login to comment 91 ABCC7 p.Cys491Ser In inside-out patch clamp recordings, C491S mutant channels show openings to two different conductance levels, with the majority of channel openings to a subconductance of ϳ3 pS (Fig. 2, Table 1). Login to comment 92 ABCC7 p.Cys491Ser In inside-out patch clamp recordings, C491S mutant channels show openings to two different conductance levels, with the majority of channel openings to a subconductance of b03;3 pS (Fig. 2, Table 1). Login to comment 93 ABCC7 p.Cys491Ser The subconductance openings of the C491S mutant are much shorter than the wild-type subconductance, as shown by comparing the dwell time histograms in Fig. 1 C with that in Fig. 2 C. Login to comment 94 ABCC7 p.Cys491Ser The subconductance openings of the C491S mutant are much shorter than the wild-type subconductance, as shown by comparing the dwell time histograms in Fig. 1 C with that in Fig. 2 C. Login to comment 95 ABCC7 p.Cys491Ser Despite this difference, the C491S subconductance openings appeared to be very similar to the wild-type subconductance in current amplitudes and in requirement for phosphorylation. Login to comment 96 ABCC7 p.Cys491Ser ABCC7 p.Cys491Ser As shown in Fig. 3, C491S mutant channels, like the wild-type subconductance, require phosphorylation by PKA for high-probability openings. Login to comment 97 ABCC7 p.Cys491Ser As shown in Fig. 3, C491S mutant channels, like the wild-type subconductance, require phosphorylation by PKA for high-probability openings. Login to comment 98 ABCC7 p.Cys491Ser While the C491S single mutation had occasional full-size openings along with subconductance openings, mutating both cysteine residues in NBD1 resulted in a channel that almost never opened to the full-size conductance (Table 1; Fig. 4, A and B). Login to comment 99 ABCC7 p.Cys491Ser While the C491S single mutation had occasional full-size openings along with subconductance openings, mutating both cysteine residues in NBD1 resulted in a channel that almost never opened to the full-size conductance (Table 1; Fig. 4, A and B). Login to comment 104 ABCC7 p.Cys491Ser As shown by the dwell time histograms in Fig. 4 C, the long component of the open time distributions for the C491/524S and C-QUAD-S mutants is much shorter than the wild-type subconductance, and closer to that of the C491S mutant. Login to comment 105 ABCC7 p.Cys524Ser ABCC7 p.Cys524Ser ABCC7 p.Cys491Ser ABCC7 p.Cys491Ser Mutation of C524 has little effect on channel gating While CFTR channels carrying the C491S mutation either alone or in combination with C524S or C1344/1355S open almost exclusively to a 3 pS subconductance, channels carry- ing only the C524S mutation exhibit conductance similar to wild-type channels. Login to comment 106 ABCC7 p.Cys524Ser ABCC7 p.Cys524Ser ABCC7 p.Cys524Ser ABCC7 p.Cys491Ser Like the wild-type channel, nearly every patch of C524S channels gates to the full-size openings, although, like wild-type channels, subconductance openings do appear (Table 1). Login to comment 107 ABCC7 p.Cys524Ser ABCC7 p.Cys524Ser Moreover, gating of the C524S mutant is sensitive to redox potential in a manner almost identical to that reported in wild-type channels, with the channel openings shortened by reducing conditions and oxidizing conditions resulting in long "locked open" bursts of the channel (Harrington et al., 1999). Login to comment 108 ABCC7 p.Cys524Ser Moreover, gating of the C524S mutant is sensitive to redox potential in a manner almost identical to that reported in wild-type channels, with the channel openings shortened by reducing conditions and oxidizing conditions resulting in long "locked open" bursts of the channel (Harrington et al., 1999). Login to comment 117 ABCC7 p.Cys491Ser In the presence of a mixture of ATP and ATP␥S, full-size channels are "locked open": opening in long bursts that can last for minutes (Anderson et al., 1991b; Baukrowitz et al., FIGURE 2 C491S-CFTR channels open most frequently to a 3-pS subconductance. Login to comment 118 ABCC7 p.Cys491Ser ABCC7 p.Cys491Ser (A) One-minute sample traces of inside-out patches from HEK 293 cells expressing the C491S mutant of CFTR showing the two modes of gating of the channel. Login to comment 119 ABCC7 p.Cys491Ser (A) One-minute sample traces of inside-out patches from HEK 293 cells expressing the C491S mutant of CFTR showing the two modes of gating of the channel. Login to comment 122 ABCC7 p.Cys491Ser (C) Open dwell time histogram of 6000 opening and closing events of C491S channels in nine separate patches. Login to comment 123 ABCC7 p.Cys491Ser (C) Open dwell time histogram of 6000 opening and closing events of C491S channels in nine separate patches. Login to comment 124 ABCC7 p.Cys491Ser (D) Current-voltage relationship of subconductance channels from C491S CFTR. Login to comment 125 ABCC7 p.Cys491Ser (D) Current-voltage relationship of subconductance channels from C491S CFTR. Login to comment 131 ABCC7 p.Cys491Ser Two other mutants (C491S, C491/524S) were tested for the effect of ATP␥S on subconductance openings (Fig. 5 C). Login to comment 132 ABCC7 p.Cys491Ser ABCC7 p.Cys491Ser Both the C491S and C491/524S mutants have open dwell times that are shorter than wild-type subconductance, and these dwell times were not significantly altered by the presence of ATP␥S (p Ͼ 0.05; Kolmogorov-Smirnov). Login to comment 133 ABCC7 p.Cys491Ser Both the C491S and C491/524S mutants have open dwell times that are shorter than wild-type subconductance, and these dwell times were not significantly altered by the presence of ATPॹS (p b0e; 0.05; Kolmogorov-Smirnov). Login to comment 134 ABCC7 p.Cys491Ser This is in contrast FIGURE 3 Increased phosphorylation by PKA increases the frequency of subconductance openings in C491S patches. Login to comment 135 ABCC7 p.Cys491Ser ABCC7 p.Cys491Ser (A) One-minute sample traces of an inside-out patch from an HEK 293 cell expressing C491S mutant CFTR with and without the addition of PKA. Login to comment 136 ABCC7 p.Cys491Ser (A) One-minute sample traces of an inside-out patch from an HEK 293 cell expressing C491S mutant CFTR with and without the addition of PKA. Login to comment 138 ABCC7 p.Cys524Ser ABCC7 p.Cys491Ser The patch was held at 75 mV. (B) Graph of open probability versus time for the patch shown in A. TABLE 1 Mutations of cysteine residues in NBD1 increase the proportion of patches with subconductance openings, while mutations of cysteines in NBD2 decrease the proportion of patches with subconductance openings Mutation Patches with Subconductance Patches with Full Size Total Patches Percent with Subconductance Percent with Full Size Wild-type 34 49 49 69 100 C491S 16 5 18 89 28 C491/524S 10 1 13 77 8 C524S 2 5 6 33 83 C1344/1355S 8 30 30 27 100 C-QUAD-S 19 0 30 63 0 The number of inside-out patches containing frequent full-size and/or subconductance channel openings were counted. Login to comment 140 ABCC7 p.Cys524Ser ABCC7 p.Cys491Ser TABLE 1 Mutations of cysteine residues in NBD1 increase the proportion of patches with subconductance openings, while mutations of cysteines in NBD2 decrease the proportion of patches with subconductance openings Mutation Patches with Subconductance Patches with Full Size Total Patches Percent with Subconductance Percent with Full Size Wild-type 34 49 49 69 100 C491S 16 5 18 89 28 C491/524S 10 1 13 77 8 C524S 2 5 6 33 83 C1344/1355S 8 30 30 27 100 C-QUAD-S 19 0 30 63 0 The number of inside-out patches containing frequent full-size and/or subconductance channel openings were counted. Login to comment 143 ABCC7 p.Cys491Ser ABCC7 p.Cys491Ser Moreover, the presence of ATP␥S shortens the open dwell time of wild-type channels to approximately that of channels containing the C491S mutant, suggesting that the altered behavior of the C491S channel may be related to changes in ATP hydrolysis at NBF1. Login to comment 145 ABCC7 p.Cys491Ser ABCC7 p.Cys491Ser Moreover, the presence of ATPॹS shortens the open dwell time of wild-type channels to approximately that of channels containing the C491S mutant, suggesting that the altered behavior of the C491S channel may be related to changes in ATP hydrolysis at NBF1. Login to comment 181 ABCC7 p.Cys491Ser In some cases the channel transitions to the subconductance without appearing to close, while sometimes it opens directly to the subconductance state and then transitions to the full-size channel. This type of rapid transition between full-size and subconductance was not observed in recordings of wild-type channels in reducing conditions, nor in the C491S, C491/524S, or C- QUAD-S mutants in which the subconductance state makes up the majority of channel openings observed. Login to comment 183 ABCC7 p.Cys491Ser ABCC7 p.Cys491Ser Effect of mutation of C1344 and C1355 on subconductance frequency and redox sensitivity Mutation of cysteine residues in the second nucleotide binding domain by themselves had much less of an effect on channel gating than the C491S mutation. Login to comment 185 ABCC7 p.Cys491Ser Effect of mutation of C1344 and C1355 on subconductance frequency and redox sensitivity Mutation of cysteine residues in the second nucleotide binding domain by themselves had much less of an effect on channel gating than the C491S mutation. Login to comment 204 ABCC7 p.Cys524Ser Mutation of C491 in NBD1 to serine resulted in channels that opened almost exclusively to a 3-pS subconductance, while C524S mutant channels showed mostly full-size openings. Login to comment 206 ABCC7 p.Cys524Ser ABCC7 p.Cys491Ser The 3-pS subconductance of channels containing the C491S mutant alone or with other cysteine mutations was similar to the subconductance observed in recordings from patches containing wild-type CFTR channels, although with a shortened open dwell time. Login to comment 208 ABCC7 p.Cys491Ser ABCC7 p.Cys491Ser The effect of the C491S mutation on control of channel conductance (enriching for subconductance at the expense of full-size openings) was unexpected; however, the specificity of this mutation for that effect is supported by several pieces of data. Login to comment 210 ABCC7 p.Cys491Ser The effect of the C491S mutation on control of channel conductance (enriching for subconductance at the expense of full-size openings) was unexpected; however, the specificity of this mutation for that effect is supported by several pieces of data. Login to comment 221 ABCC7 p.Cys491Ser With channels containing the C491S mutation opening almost exclusively to a subconductance with gating properties very different from the full-size channel, it is difficult to directly relate the effect of redox potential on gating kinetics of the mutants compared to the wild-type channel. Login to comment 223 ABCC7 p.Cys491Ser With channels containing the C491S mutation opening almost exclusively to a subconductance with gating properties very different from the full-size channel, it is difficult to directly relate the effect of redox potential on gating kinetics of the mutants compared to the wild-type channel. Login to comment 244 ABCC7 p.Cys491Ser Our data suggest that subconductance opening may be related to a decreased hydrolysis by the first nucleotide binding domain because mutation of C491S in NBD1 results in an increase in subconductance frequency at the expense of the full-size openings. Recently published work with truncation mutants of CFTR has suggested that the CFTR channel might be "double-barreled," with one pore producing the full-size conductance and a second, independently gated pore producing a 3-4-pS subconductance (Yue et al., 2000). Login to comment 247 ABCC7 p.Cys491Ser Our data suggest that subconductance opening may be related to a decreased hydrolysis by the first nucleotide binding domain because mutation of C491S in NBD1 results in an increase in subconductance frequency at the expense of the full-size openings. Recently published work with truncation mutants of CFTR has suggested that the CFTR channel might be "double-barreled," with one pore producing the full-size conductance and a second, independently gated pore producing a 3-4-pS subconductance (Yue et al., 2000). Login to comment 256 ABCC7 p.Cys491Ser The frequency of subconductance openings in CFTR channels with the C491S mutation may mean that this region of the molecule is important for the type of intermolecular interactions observed by other groups to be important for high-frequency opening of the full-size channel. Login to comment 259 ABCC7 p.Cys491Ser The frequency of subconductance openings in CFTR channels with the C491S mutation may mean that this region of the molecule is important for the type of intermolecular interactions observed by other groups to be important for high-frequency opening of the full-size channel. It is conceivable that the cysteine residues in NBD1, particularly C491, could be important in the intermolecular associations that stabilize a dimer of the channel and allow it to produce full-size openings. Login to comment