Proton/hydroxyl permeability of proximal tubule brush border vesicles

Abstract

The net H+/OH- permeability of rabbit renal proximal tubule brush border membrane vesicles was determined by measuring the rate of collapse of preformed pH gradients using acridine orange. The membranes were voltage clamped using valinomycin and [K+]in = [K+]out. Internal buffer capacity was determined by titration of lysed vesicles and by titration of measured Na+/H+ exchange rates with exogenously added buffers. Both methods revealed an intravesicular buffer capacity of 125-135 mM/pH unit at pH 6.0 and 20 degrees C. Using this buffer capacity, the net H+/OH- permeability was found to be 5 X 10(-3) cm/s in brush border vesicles prepared by Mg2+ aggregation. The rate of collapse of pH gradients in brush border vesicles prepared by sucrose density gradient centrifugation was virtually identical to the rate in vesicles prepared with Mg2+, indicating that the high H+/OH- permeability was not an artifact of Mg2+ preparation. Activation energy of the H+/OH- permeability pathway was 4.9 kcal/mol, whereas activation energy of the Na+/H+ antiporter was 11.4 kcal/mol. Since the rate of H+/OH- diffusion was not affected by amiloride, it is concluded that H+/OH- permeate through brush border membranes by a pathway separate from the Na+/H+ antiporter. This pathway is not inhibited by dicyclohexylcarbodiimide at concentrations up to 2 mM but is inhibited by 0.2-5 mM p-chloromercuribenzenesulfonate, suggesting the presence of a sulfhydryl group in the pathway.(ABSTRACT TRUNCATED AT 250 WORDS)