Polarized apical-membrane expression of cAMP-actlvated chloride channels in isolated epithelial cells

We tested the hypothesis that cAMP-stimulated CI channels are expressed exclusively on the apical membrane of isolated polarized cells (figure-eight cells) from Necturus gallbladder (NGB) epithelium. In the intact NGB epithelium, the electrophysiological characteristics of these channels are similar, but not identical, to those of mammalian CFTR, suggesting expression of a CFTR homologue. Degenerate oligonucleotides were used to amplify the R-domain of CFTR. The 700-bp amplified fragment had a deduced amino-acid sequence 67% identical to that of the human R-domain. This fragment was used as a probe in a Northern blot of NGB epithelium, yielding a positive band of ca. 5 kb. Immunofluorescence with an anti-human CFTR antibody in figureeight cells demonstrated labelling of the apical-, but not the basolateralmembrane domain. Intracellular-microelectrode studies in figure-eight cells demonstrated "normal" membrane voltage (ca. -65 mV) and a ca. 20-mV depolarization with forskolin. Regional superfusion experiments with high-Kand low-CI solutions revealed high apical and basolateral Kconductance (GK), and a small basolateral GCI, under control conditions. After forskolin, the conductance was dominated by an apical-membrane GC!. Whole-cell patchclamp studies yielded a forskolin-activated linear CI' current, with high selectivity for CIover aspartate. These results demonstrate polarized expression of a cAMP-activated CI channel in figure-eight cells from NGB epithelium and suggest that this channel is a CFTR homologue. [Supported by NIH Grants DK-38734 and DK-40701, Veterans Administration and Cystic Fibrosis Foundation.].

Duke Scholars

Author Jonathan Allen Cohn Medicine, Gastroenterology