Mechanism of coupling between Cl − and OH − transport in renal brush-border membranes

Abstract

The coupling mechanism for Cl − and H +/OH − transport in renal brush-border vesicles was examined from intravesicular pH changes following imposed H + and Cl − gradients. Vesicles were loaded with 6-carboxyfluorescein and exposed to H + gradients and Cl −, gluconate, or sulfate gradients, each with and without a K +/valinomycin voltage clamp. Parallel experiments were performed with vesicles equilibrated with 10 mM HCO 3 − or 5 mM formate. Rate of H +/OH − transport was determined from the initial rate of change in 6-carboxyfluorescein fluorescence, vesicle buffer capacity and the relationship between fluorescence and vesicle pH. In contrast to gluconate or sulfate, Cl − caused enhanced H +/OH − transport under all conditions. This difference was eliminated with voltage clamping in the presence of gluconate, SO 4 2−, or HCO 3 −, but not in the presence of formate. These findings were not affected by the method of preparation of the vesicles. Electrically coupled Cl −/OH − transport was not inhibited by 100 μM DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonate) or 100 μM DBDS (4,4′-dibenzamidostilbene-2,2′-disulfonate). SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate) was found to be a protonphore at concentrations > 500 μM. As a control for the method, we demonstrated amiloride inhibitable, electroneutral Na +-H + exchange (H + flux = 107 ± 9 nmol/s per mg, 100 mM Na +) and electroneutral, DBDS inhibitable Cl −-HCO 3 − exchange in sealed human red blood cell ghosts. Therefore, electroneutral Cl −-OH − or HCO 3 − exchange does not measurably contribute to Cl − transport in the proximal tubule brush border. Cl −-formate exchange with formic acid recyling appears to be the only electroneutral coupling mechanism between Cl − and OH − transport demonstrable in renal brush-border membrane vesicles.