Calcium-activated chloride currents in primary cultures of rabbit distal convoluted tubule

Chloride (Cl-) conductances were studied in primary cultures of rabbit distal convoluted tubule (very early distal ''bright'' convoluted tubule, DCTb) by the whole cell patch-clamp technique. We identified a Cl- current activated by 2 mu M extracellular ionomycin. The kinetics of the macroscopic current were time dependent for depolarizing potentials with a slow developing component. The steady-state current presented outward rectification, and the ion selectivity sequence was I- > Br- much greater than Cl- > glutamate. The current was inhibited by 0.1 mM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and 1 mM diphenylamine-2-carboxylate. To identify the location of the Cl- conductance, 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence experiments were carried out in confluent cultures developed on collagen-coated permeable filters. Cl- removal from the apical solution induced a Cl- efflux that was stimulated by 10 mu M forskolin. Forskolin had no effect on the basolateral Cl- permeability. Cl- substitution in the basolateral solution induced an efflux stimulated by 2 mu M ionomycin or 50 mu M extracellular ATP. Ionomycin had no effect on the apical Cl- fluxes. Thus cultured DCTb cells exhibit Ca2+-activated Cl- channels located in the basolateral membrane. This Cl- permeability was active at a resting membrane potential and could participate in the Cl- reabsorption across the DCTb in control conditions.