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Volume 271, Number 44, Issue of November 1, 1996 pp. 27493-27499
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Cytoplasmic Loop Three of Cystic Fibrosis Transmembrane Conductance Regulator Contributes to Regulation of Chloride Channel Activity

(Received for publication, May 3, 1996, and in revised form, August 22, 1996)

From the Medical Research Council Group in Membrane Biology, Departments of Medicine and Biochemistry, University of Toronto, Toronto, Ontario, Canada, M5S 1A8, the ^§ Department of Physiology, McGill University, Montreal, Quebec, Canada, H3G 1Y6, and the ^'' Mayo Graduate School of Medicine and Department of Biochemistry and Molecular Biology, S. C. Johnson Medical Research Center, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259

To examine the contribution of the large cytoplasmic loops of the cystic fibrosis transmembrane conductance regulator (CFTR) to channel activity, the three point-mutations (S945L, H949Y, G970R) were characterized that have been detected in the third cytoplasmic loop (CL3, residues 933-990) in patients with cystic fibrosis. Chinese hamster ovary cell lines stably expressing wild-type CFTR or mutant G970R-CFTR yielded polypeptides with apparent masses of 170 kDa as the major products, whereas the major products of mutants S945L-CFTR and H949Y-CFTR had apparent masses of 150 kDa. The 150-kDa forms of CFTR were sensitive to endoglycosidase H digestion, indicating that these mutations interfered with maturation of the protein. Increased levels of mature CFTR (170 kDa) could be obtained for mutant H949Y when cells were grown at a lower temperature (26 °C) or incubated in the presence of 10% glycerol. For all mutants, the open probability (P₀) of the CFTR channels was significantly altered. S945L-CFTR and G970R-CFTR showed a severe reduction in the P₀, whereas the H949Y mutation doubled the P₀ relative to wild-type. The changes in P₀ predominantly resulted from an alteration of the mean burst durations which suggests that CL3 is involved in obtaining and/or maintaining stability of the open state. In addition, mutants S945L and G970R had current-voltage relationships that were not completely linear over the range ±80 mV, but showed slight outward rectification. The fact that CL3 mutations can have subtle effects on channel conductance indicates that this region may be physically close to the inner mouth of the pore.

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