ABCMdb

PMID: 9920885

Lee MG, Wigley WC, Zeng W, Noel LE, Marino CR, Thomas PJ, Muallem S
Regulation of Cl-/ HCO3- exchange by cystic fibrosis transmembrane conductance regulator expressed in NIH 3T3 and HEK 293 cells.
J Biol Chem. 1999 Feb 5;274(6):3414-21., 1999-02-05 [PubMed]

Sentences

No. Mutations Sentence Comment

52 ABCC7 p.Pro205Ser ABCC7 p.Asp1370Asn ABCC7 p.Gly1249Glu ABCC7 p.Lys1250Met ABCC7 p.Gly1247Asp The mutagenesis primers were as follows: P205S primer, 5Ј-CGT GTG GAT CGC TTC TTT GCA AGT GGC-3Ј; W846term, 5Ј-GAG CAT ACC AGC AGT GAC TAC ATA GAA CAC ATA CCT TCG ATA TAT TAC-3Ј; G1247D/G1249E, 5Ј-GTG GGC CTC TTG GGA AGA ACT GAT TCA GAG AAG AGT ACT TTG TTA TCA GC-3Ј; K1250M, 5Ј-CTT GGG AAG AAC TGG ATC AGG GAT GAG TAC TTT GTT ATC AGC-3Ј; D1370N, 5Ј-GTA AGG CGA AGA TCT TGC TGC TTA ATG AAC CCA GTG CTC ATT TGG ATC-3Ј. Login to comment 161 ABCC7 p.Pro205Ser Fig. 6, c and d shows the localization of WT CFTR relative to that of BiP, and g and h show the localization of P205S CFTR. Login to comment 162 ABCC7 p.Pro205Ser It is clear that WT CFTR was present in the ER and plasma membrane whereas P205S CFTR localized in the ER. Login to comment 163 ABCC7 p.Asp1370Asn ABCC7 p.Gly1249Glu ABCC7 p.Lys1250Met ABCC7 p.Gly1247Asp Fig. 6 (i-k) shows the plasma membrane localization of K1250M CFTR, D1370N CFTR, and the double mutant G1247D/ G1249E CFTR, respectively. Login to comment 185 ABCC7 p.Pro205Ser Panels e and g show the predominant ER localization of ⌬F508 and P205S CFTR, respectively. Login to comment 234 ABCC7 p.Pro205Ser ⌬F508 and P205S CFTR are known maturation mutants (26, 27) that do not reach the plasma membrane of 293 cells (Fig. 6). Login to comment 239 ABCC7 p.Gly1249Glu ABCC7 p.Gly1247Asp For example, the G1247D/G1249E CFTR double mutant was reported to have no Cl-channel activity (32), was expressed in the plasma membrane (Fig. 6k), and had no effect on AE activity (Fig. 12a). Login to comment 240 ABCC7 p.Lys1250Met The K1250M CFTR mutant had increased channel activity (32), was expressed in the plasma membrane (Fig. 6i) and activated AE similar to WT CFTR (Fig. 12b). Login to comment 241 ABCC7 p.Asp1370Asn However, D1370N CFTR had nearly normal Cl-channel activity (32) and was expressed in the plasma membrane (Fig. 6j), but was unable to activate AE (Fig. 12c). Login to comment 256 ABCC7 p.Pro205Ser Folding CFTR mutants and N-terminal half CFTR had no effect on AE activity. HEK 293 cells were cotransfected with plasmids carrying GFP and P205S CFTR (a), ⌬F508 CFTR (b), or Trp-846 termination codon CFTR (c). Login to comment 264 ABCC7 p.Gly1249Glu ABCC7 p.Gly1247Asp The G1247D/G1249E double mutant was expressed in the plasma membrane but had no effect on AE activity (a). Login to comment 265 ABCC7 p.Lys1250Met The K1250M mutant was at least as effective as WT CFTR in stimulating AE activity (b). Login to comment 266 ABCC7 p.Asp1370Asn The D1370N mutant had minimal effect on AE activity (c). Login to comment 272 ABCC7 p.Pro205Ser This is further supported by the findings with the ⌬F508 and P205S CFTR maturation mutants, which showed that expression of CFTR in the plasma membrane, rather than mere expression of CFTR in the cells, was required for activation of AE. Login to comment 273 ABCC7 p.Asp1370Asn In this respect the results obtained with D1370N CFTR are of particular interest since this mutation in NBD2 did not ablate channel activity (32) but eliminated regulation of AE activity by CFTR. Login to comment 274 ABCC7 p.Asp1370Asn Recently, it was reported that the D1506A mutation of the sulfonylurea receptor 1 protein, which corresponds to D1370N of human CFTR, similarly failed to stimulate the KATP channel (33). Login to comment