ABCMdb

PMID: 9325282

Cotten JF, Welsh MJ
Covalent modification of the regulatory domain irreversibly stimulates cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 1997 Oct 10;272(41):25617-22., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC7 p.Cys832Ala 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 ABCC7 p.Cys832Ala 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 ABCC7 p.Cys832Ala 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 80 ABCC7 p.Phe1450* NEM Stimulates in Part through R Domain Modification-To identify the cysteine(s) through which NEM was mediating its stimulatory effect, we examined the NEM response of CFTR variants in which individual or groups of cysteines were mutated to alanine(s) or deleted (F1450X deletes Cys1458 ). Login to comment 81 ABCC7 p.Phe1450* NEM Stimulates in Part through R Domain Modification-To identify the cysteine(s) through which NEM was mediating its stimulatory effect, we examined the NEM response of CFTR variants in which individual or groups of cysteines were mutated to alanine(s) or deleted (F1450X deletes Cys1458 ). Login to comment 82 ABCC7 p.Cys832Ala However one mutant, CFTR-C832A, showed a marked decrease in its response to NEM (Fig. 4). Login to comment 83 ABCC7 p.Cys832Ala However one mutant, CFTR-C832A, showed a marked decrease in its response to NEM (Fig. 4). Login to comment 124 ABCC7 p.Phe1450* Effect of NEM on CFTR containing cysteine to alanine mutations, on CFTR-⌬708-835, and on CFTR in which a portion of the C terminus has been deleted (F1450X). Login to comment 125 ABCC7 p.Phe1450* Effect of NEM on CFTR containing cysteine to alanine mutations, on CFTR-D708-835, and on CFTR in which a portion of the C terminus has been deleted (F1450X). Login to comment 146 ABCC7 p.Cys832Ala We also examined the effect of NEM on the single channel properties of CFTR-C832A. Login to comment 147 ABCC7 p.Cys832Ala We also examined the effect of NEM on the single channel properties of CFTR-C832A. Login to comment 148 ABCC7 p.Cys832Ala 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 149 ABCC7 p.Cys832Ala 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 ABCC7 p.Cys832Ala The behavior of CFTR-C832A was very similar to that of wild-type protein (Fig. 6B). Login to comment 151 ABCC7 p.Cys832Ala The behavior of CFTR-C832A was very similar to that of wild-type protein (Fig. 6B). Login to comment 152 ABCC7 p.Cys832Ala Like wild-type CFTR, Po and burst duration decreased when PKA was removed from CFTR-C832A. Login to comment 153 ABCC7 p.Cys832Ala Like wild-type CFTR, Po and burst duration decreased when PKA was removed from CFTR-C832A. Login to comment 154 ABCC7 p.Cys832Ala 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 155 ABCC7 p.Cys832Ala 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 ABCC7 p.Cys832Ala Single channel traces of wild-type CFTR (A) and CFTR-C832A (B) studied in excised, inside-out membrane patches. Login to comment 169 ABCC7 p.Cys832Ala Single channel traces of wild-type CFTR (A) and CFTR-C832A (B) studied in excised, inside-out membrane patches. Login to comment 173 ABCC7 p.Cys832Ala 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 174 ABCC7 p.Cys832Ala 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 176 ABCC7 p.Cys832Ala 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 ABCC7 p.Cys832Ala ABCC7 p.Cys832Ala 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 178 ABCC7 p.Cys832Ala 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 ABCC7 p.Cys832Ala 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 181 ABCC7 p.Cys832Ala 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