AJP - Renal Physiology, Vol 248, Issue 1 78-F86, Copyright © 1985 by American Physiological Society
Proton/hydroxyl permeability of proximal tubule brush border vesicles
H. E. Ives
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)
