ABCMdb

ABCC7 p.Cys832Ala

None has been submitted yet.

Publications

PMID: 15623556 [PubMed] Berger AL et al: "Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain."

No. Sentence Comment

47 Cys insertion and the ''WT control`` (Fig. 5) also contained the C832A substitution, which prevents NEM modi- Abbreviations: NBD, nucleotide-binding domain; NEM, N-ethylmaleimide. Login to comment

PMID: 20133716 [PubMed] Wang W et al: "ATP-independent CFTR channel gating and allosteric modulation by phosphorylation."

No. Sentence Comment

211 For the MTS (methanethiosulfonate) experiments (Fig. S2), the cysteine substitutions (e.g., K978C) were introduced into the C832A background because modification of C832 can affect CFTR currents (34). Login to comment

PMID: 20952391 [PubMed] Wang G et al: "State-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gating by a high affinity Fe3+ bridge between the regulatory domain and cytoplasmic loop 3."

No. Sentence Comment

123 NEM doubled hCFTR activity (Fig. 3A), but C832A clearly stopped potentiation (Fig. 3B). Login to comment
128 Fig. 3, E and F, further supports this proposal because C832A weakened inhibition by Fe3ϩ . Login to comment
129 This weak binding affinity seen with C832A was not due to a high open probability because the C832A mutant channel was further activated by curcumin dramatically (Fig. 3E). Login to comment
145 In support of this proposal, H950A/H954A and D836A/C832A/ H774A completely prevented Fe3ϩ inhibition, which was reversed by EDTA (Fig. 4E). Login to comment
170 Macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing the hCFTR (A and C) and the C832A mutant (B and E). Login to comment
171 Effects of NEM were tested on the hCFTR channel (A and C) and the C832A mutant (B). Login to comment
174 E, effect of Fe3ϩ on the C832A mutant. Login to comment
175 F, fractional Fe3ϩ inhibition of the current for WT and C832A (n ϭ 4-9). Login to comment

PMID: 9822656 [PubMed] Cotten JF et al: "Covalent modification of the nucleotide binding domains of cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

37 1 The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; NEM, N-ethylmaleimide; PKA, catalytic subunit of cAMP-dependent protein kinase; ABC, ATP-binding cassette; NBD, nucleotide binding domain; NBD1-Cys, A462C/C832A; NBD2-Cys, C832A/S1248C; TB, mean burst duration; g, single channel conductance; ␶cs, slow, long closed time interval; ␶o, open time interval; ␶, time constant for rate of NEM modification; Iϱ, percentage of current remaining following complete NEM modification; TES, N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid. Login to comment
73 NEM inhibits NBD1-Cys (CFTR-A462C/C832A) and NBD2-Cys(CFTR-S1248C/C832A) channel activity in an ATP-dependent manner. A, CFTR-C832A; B, CFTR-NBD1-Cys; C, CFTR-NBD2-Cys. Login to comment
85 Both NBD1-Cys and NBD2-Cys mutants also contain the C832A mutation. Login to comment
87 To determine the functional consequences of NEM modification of the NBDs, we applied 100 ␮M NEM to the cytoplasmic side of excised membrane macropatches containing CFTR-C832A (control), NBD1-Cys, or NBD2-Cys mutant channels. Login to comment
88 As we have shown previously (33), 100 ␮M NEM had a slight stimulatory effect on CFTR-C832A channel activity (Fig. 2A). Login to comment
102 The NEM response of the CFTR-C832A mutant is also quantified in Fig. 3B. Login to comment
118 Our earlier work showed that NEM did not alter the single channel properties of CFTR-C832A (33). Login to comment
129 B, percentage of current remaining 5 min after application of 100 ␮M NEM for CFTR-C832A (control) and NBD1-Cys mutants. Login to comment

PMID: 9325282 [PubMed] Cotten JF et al: "Covalent modification of the regulatory domain irreversibly stimulates cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

6 At the single channel level, CFTR in which Cys832 was mutated to alanine behaved identically to wild-type CFTR, except that it failed to respond to NEM. Login to comment
58 Similar results were obtained with a value of 10 or 30 ms for both wild-type CFTR and CFTR-C832A (Ϯ PKA). Login to comment
59 Regions of data from multichannel patches where there were no superimposed openings were used for burst analysis for the CFTR-C832A mutant following PKA washout, as described previously (16). Login to comment
82 However one mutant, CFTR-C832A, showed a marked decrease in its response to NEM (Fig. 4). Login to comment
146 We also examined the effect of NEM on the single channel properties of CFTR-C832A. Login to comment
148 First, if the CFTR-C832A mutation produced a channel with a very high Po (e.g. nearing a value of 1.0), then it might appear to be resistant to NEM because Po was already near maximal. Login to comment
150 The behavior of CFTR-C832A was very similar to that of wild-type protein (Fig. 6B). Login to comment
152 Like wild-type CFTR, Po and burst duration decreased when PKA was removed from CFTR-C832A. Login to comment
154 These data indicate that CFTR-C832A is functionally and structurally similar to wild-type CFTR; however, the failure to respond to NEM identifies Cys832 as an important site of modification. Login to comment
168 Single channel traces of wild-type CFTR (A) and CFTR-C832A (B) studied in excised, inside-out membrane patches. Login to comment
173 TABLE I Single channel data for wild-type CFTR and CFTR-C832A Data were collected with membrane voltage clamped at -80 mV in the presence of 1 mM ATP. Login to comment
176 For wild-type CFTR, the average number of events used to calculate time constants for each experimental condition in each individual experiment was 1039, and for CFTR-C832A it was 775. Login to comment
177 The average number of bursts used at each experimental condition in each experiment to calculate mean burst duration was 337 and 297 for wild-type and CFTR-C832A, respectively. Login to comment
180 n ATP ϩ PKA ATP ATP ϩ NEM Wild-type CFTR Po 11 0.51 Ϯ 0.02 0.38 Ϯ 0.02a 0.57 Ϯ 0.02b g (pS) 8 ND 9.6 Ϯ 0.4 10 Ϯ 0.6 TB (ms) 5 184 Ϯ 16 149 Ϯ 8a 237 Ϯ 17b ␶o (ms) 5 68 Ϯ 7 58 Ϯ 7 56 Ϯ 7 ␶cf (ms) 5 2.16 Ϯ 0.09 2.37 Ϯ 0.11 2.39 Ϯ 0.15 ␶cs (ms) 5 141 Ϯ 25 152 Ϯ 12 118 Ϯ 8b CFTR-C832A Po 7-9 0.55 Ϯ 0.03 0.39 Ϯ 0.02a 0.42 Ϯ 0.04 g (pS) 6-8 ND 10.2 Ϯ 0.3 10.3 Ϯ 0.2 TB (ms) 3-5 194 Ϯ 19 130 Ϯ 10a 133 Ϯ 10 ␶o (ms) 3-5 62 Ϯ 3 51 Ϯ 5 50 Ϯ 4 ␶cf (ms) 3-5 2.07 Ϯ 0.13 2.63 Ϯ 0.37 2.18 Ϯ 0.28 ␶cs (ms) 3 164 Ϯ 46 206 Ϯ 75 185 Ϯ 81 a p Ͻ 0.05 relative to CFTR in the presence of PKA and ATP. Login to comment
83 However one mutant, CFTR-C832A, showed a marked decrease in its response to NEM (Fig. 4). Login to comment
147 We also examined the effect of NEM on the single channel properties of CFTR-C832A. Login to comment
149 First, if the CFTR-C832A mutation produced a channel with a very high Po (e.g. nearing a value of 1.0), then it might appear to be resistant to NEM because Po was already near maximal. Login to comment
151 The behavior of CFTR-C832A was very similar to that of wild-type protein (Fig. 6B). Login to comment
153 Like wild-type CFTR, Po and burst duration decreased when PKA was removed from CFTR-C832A. Login to comment
155 These data indicate that CFTR-C832A is functionally and structurally similar to wild-type CFTR; however, the failure to respond to NEM identifies Cys832 as an important site of modification. Login to comment
169 Single channel traces of wild-type CFTR (A) and CFTR-C832A (B) studied in excised, inside-out membrane patches. Login to comment
174 TABLE I Single channel data for wild-type CFTR and CFTR-C832A Data were collected with membrane voltage clamped at 280 mV in the presence of 1 mM ATP. Login to comment
178 The average number of bursts used at each experimental condition in each experiment to calculate mean burst duration was 337 and 297 for wild-type and CFTR-C832A, respectively. Login to comment
181 n ATP 1 PKA ATP ATP 1 NEM Wild-type CFTR Po 11 0.51 6 0.02 0.38 6 0.02a 0.57 6 0.02b g (pS) 8 ND 9.6 6 0.4 10 6 0.6 TB (ms) 5 184 6 16 149 6 8a 237 6 17b to (ms) 5 68 6 7 58 6 7 56 6 7 tcf (ms) 5 2.16 6 0.09 2.37 6 0.11 2.39 6 0.15 tcs (ms) 5 141 6 25 152 6 12 118 6 8b CFTR-C832A Po 7-9 0.55 6 0.03 0.39 6 0.02a 0.42 6 0.04 g (pS) 6-8 ND 10.2 6 0.3 10.3 6 0.2 TB (ms) 3-5 194 6 19 130 6 10a 133 6 10 to (ms) 3-5 62 6 3 51 6 5 50 6 4 tcf (ms) 3-5 2.07 6 0.13 2.63 6 0.37 2.18 6 0.28 tcs (ms) 3 164 6 46 206 6 75 185 6 81 a p , 0.05 relative to CFTR in the presence of PKA and ATP. Login to comment