Demonstration of an apical chloride conductive pathway in granular cells of toad urinary bladder

Chloride electrodiffusion across the apical membrane of granular cells from toad urinary bladder, an analogue of mammalian principal cells, was examined using the patch clamp technique. A chloride conductance was demonstrated in cell-attached membrane patches exposed to barium chloride pipette solutions. A change in the pipette chloride concentration from 30 to 100 mM caused a shift in the current voltage curve which demonstrated chloride selectivity. The chloride conductance was also examined in excised, inside out membrane patches using choline chloride solutions (chloride:choline selectivity ratio was 18:1). A closed and two open chloride conductive states were found (states A and B, 10.1 +/- 1.0 and 17.2 +/- 5.5 pS, respectively, p < 0.01). Incubation of the preparation with arginine vasopressin, dibutyryl-cAMP, or 8-bromo-cAMP approximately doubled chloride conductance to 16.6 +/- 1.7 pS (p < 0.01). The enhanced electrodiffusion was accounted for by a shift in the channel kinetics from the closed state C to the high conductance state B (p < 0.05, n = 9). 4,4'-Diisothio-cyanatostilbene- 2,2'-disulfonic acid (DIDS) and 9-anthracene-carboxylic acid (9-AC) failed to block the chloride currents. In conclusion, the regulated apical chloride conductance described would balance the sodium and potassium electrodiffusive pathways and maintain a stable membrane potential, facilitating overall conductive transport by these cells.