Disease-associated mutations in the fourth cytoplasmic loop of cystic fibrosis transmembrane conductance regulator compromise biosynthetic processing and chloride channel activity

被引:109
作者
Seibert, FS
Linsdell, P
Loo, TW
Hanrahan, JW
Clarke, DM
Riordan, JR
机构
[1] MAYO CLIN SCOTTSDALE,MAYO FDN,DEPT BIOCHEM & MOLEC BIOL,SCOTTSDALE,AZ 85259
[2] MAYO CLIN SCOTTSDALE,MAYO FDN,SC JOHNSON MED RES CTR,SCOTTSDALE,AZ 85259
[3] UNIV TORONTO,MRC,GRP MEMBRANE BIOL,DEPT MED,TORONTO,ON M5S 1A8,CANADA
[4] UNIV TORONTO,MRC,GRP MEMBRANE BIOL,DEPT BIOCHEM,TORONTO,ON M5S 1A8,CANADA
[5] MCGILL UNIV,DEPT PHYSIOL,MONTREAL,PQ H3G 1Y6,CANADA
关键词
D O I
10.1074/jbc.271.25.15139
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A cluster of 18 point mutations in exon 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been detected in patients with cystic fibrosis. These mutations cause single amino acid substitutions in the most C-terminal cytoplasmic loop (CL4, residues 1035-1102) of the CFTR chloride channel, Heterologous expression of the mutants showed that 12 produced only core-glycosylated CFTR, which was retained in the endoplasmic reticulum; the other six mutants matured and reached the cell surface, In some cases substitution of one member of pairs of adjacent residues resulted in misprocessing, whereas the other did not, Thus, the secondary structure of CL4 may contribute crucially to the proper folding of the entire CFTR molecule, Cyclic AMP stimulated iodide efflux was not detected from cells expressing the misprocessed variants but was from the other six, indicating that their mutations cause relatively subtle channel defects. Consistent with this, these latter mutations generally are present in patients who are pancreatic-sufficient, while the processing mutants are mostly from patients who are pancreatic-insufficient. Single-channel patch-clamp analysis demonstrated that the processed mutants had the same ohmic conductance as wild-type CFTR, but a lower open probability, generally due to an increase in channel mean closed time and a reduction in mean open time, This suggests that mutations in CL4 do not affect pore properties of CFTR, but disrupt the mechanism of channel gating.
引用
收藏
页码:15139 / 15145
页数:7
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