ATP-dependent renal H+ translocation: regional localization, kinetic characteristics, and chloride dependence

We characterized Mg(2+)-dependent ATPase activity in membranes from the renal cortex, the outer and inner stripes of the outer medulla, and papillary vesicles. In all regions, there was Mg(2+)-dependent ATPase activity that was resistant to oligomycin and vanadate and sensitive to N,N'-dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide, and filipin. DCCD-Sensitive Mg(2+)-ATPase activity was highest in the inner stripe of the outer medulla and lowest in the cortex, with intermediate values in the outer stripe of the outer medulla and papilla. The Km for ATP, however, was similar among the different regions of the kidney. DCCD-Sensitive Mg(2+)-ATPase activity was critically dependent upon chloride with Km for Cl- in the range of 2-5 mM. In the presence of ATP, this ATPase was capable of H+ translocation, as assessed by acridine orange quenching. Inhibitors of ATPase activity prevented H+ translocation, which suggests that the Mg(2+)-ATPase represents, at least in part, an H(+)-ATPase. H+ transport was likewise critically dependent upon chloride, with similar Km. The effect of chloride on H+ translocation was blocked by the chloride channel inhibitor, diphenylamine-2 carboxylic acid. In the absence of chloride, H+ transport was abolished, but it could be partially restored by the creation of a favorable electric gradient by K+ and valinomycin. These studies demonstrate that the renal H(+)-ATPase exhibits different activities in various regions of the kidney. The ATPase activity and H+ translocation are critically dependent upon the presence of chloride, which suggests that chloride influences H+ translocation by dissipating the H+ gradient and acting at the catalytic site of the ATPase.