POLARIZED EXPRESSION OF CAMP-ACTIVATED CHLORIDE CHANNELS IN ISOLATED EPITHELIAL-CELLS

We have described a preparation of Necturus maculosus gall bladder (NG B) epithelium yielding isolated cells that retain structural and funct ional polarity (''figure-eight'' cells). These cells have a normal mem brane voltage and remain polarized for several hours after isolation. Apical and basolateral membrane domains are differentially labeled wit h hydrophobic fluorescent dyes; freeze-fracture electron microscopy re veals two distinct membrane domains separated by tight junctions; ZO-1 , Na+/H+ exchanger (NKE3), and Na+-K+-ATPase are present in the juncti onal, apical, and basolateral region, respectively; and cell-attached patch-clamp experiments reveal different K+ currents in the two membra ne domains [R. J. Torres, G. A. Altenberg, J. A. Copello, G. Zampighi, and L. Reuss, Am. J. Physiol. 270 (Cell Physiol. 39): C1864-C1874, 19 96]. Here, we show that NGB epithelial cells express a protein cross-r eactive with an antibody against human cystic fibrosis transmembrane c onductance regulator (CFTR). In figure-eight cells, immunoreactivity w as restricted to the apical membrane domain. Using intracellular micro electrodes and a novel method of regional superfusion, we found that c ontrol cells have high K+ conductances in both membranes and a small b asolateral Cl- conductance, similar to findings in the epithelium. Act ivation of adenylate cyclase with forskolin elicited a large apical me mbrane Cl- conductance and membrane depolarization. Whole cell patch-c lamp studies yielded a forskolin-activated linear Cl- current, with hi gh Cl-/aspartate selectivity. In conclusion, 1) figure-eight cells mai ntain the conductive membrane properties present in the epithelium, in cluding polarized expression of adenosine 3',5'-cyclic monophosphate ( cAMP)-activated Cl- channels, and 2) the cAMP-activated Cl- conductanc e is underlied by a CFTR homologue.