DISEASE-ASSOCIATED MUTATIONS IN THE 4TH CYTOPLASMIC LOOP OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR COMPROMISE BIOSYNTHETIC PROCESSING AND CHLORIDE CHANNEL ACTIVITY

A cluster of 18 point mutations in exon 17b of the cystic fibrosis tra nsmembrane conductance regulator (CFTR) gene has been detected in pati ents with cystic fibrosis. These mutations cause single amino acid sub stitutions in the most C-terminal cytoplasmic loop (CL4, residues 1035 -1102) of the CFTR chloride channel, Heterologous expression of the mu tants showed that 12 produced only core-glycosylated CFTR, which was r etained in the endoplasmic reticulum; the other six mutants matured an d reached the cell surface, In some cases substitution of one member o f pairs of adjacent residues resulted in misprocessing, whereas the ot her did not, Thus, the secondary structure of CL4 may contribute cruci ally to the proper folding of the entire CFTR molecule, Cyclic AMP sti mulated iodide efflux was not detected from cells expressing the mispr ocessed variants but was from the other six, indicating that their mut ations cause relatively subtle channel defects. Consistent with this, these latter mutations generally are present in patients who are pancr eatic-sufficient, while the processing mutants are mostly from patient s who are pancreatic-insufficient. Single-channel patch-clamp analysis demonstrated that the processed mutants had the same ohmic conductanc e 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 tim e, This suggests that mutations in CL4 do not affect pore properties o f CFTR, but disrupt the mechanism of channel gating.