CYTOPLASMIC LOOP 3 OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR CONTRIBUTES TO REGULATION OF CHLORIDE CHANNEL ACTIVITY

To examine the contribution of the large cytoplasmic loops of the cyst ic fibrosis transmembrane conductance regulator (CFTR) to channel acti vity, the three point-mutations (S945L, H949Y, G970R) were characteriz ed that have been detected in the third cytoplasmic loop (CL3, residue s 933-990) in patients with cystic fibrosis, Chinese hamster ovary cel l lines stably expressing wildtype CFTR or mutant G970R-CFTR yielded p olypeptides with apparent masses of 170 kDa as the major products, whe reas the major products of mutants S945L-CFTR and H949Y-CFTR had appar ent masses of 150 kDa. The 150-kDa forms of CFTR were sensitive to end oglycosidase H digestion, indicating that these mutations interfered w ith maturation of the protein, Increased levels of mature CFTR (170 kD a) could be obtained for mutant H949Y when cells were grown at a lower temperature (26 degrees C) or incubated in the presence of 10% glycer ol, For all mutants, the open probability (P-0) of the CFTR channels w as significantly altered, S945L-CFTR and G970R-CFTR showed a severe re duction in the P-0, whereas the H949Y mutation doubled the P-0 relativ e to wild type. The changes in P-0 predominantly resulted from an alte ration of the mean burst durations which suggests that CL3 is involved in obtaining and/or maintaining stability of the open state. In addit ion, mutants S945L and G970R had current-voltage relationships that we re not completely linear over the range +/-80 mV, but showed slight ou tward rectification, The fact that CL3 mutations can have subtle effec ts on channel conductance indicates that this region may be physically close to the inner mouth of the pore.