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Abstract

Exposure of porcine renal brush-border membrane vesicles to 1.2% cholate and subsequent detergent removal by dialysis reorients almost all N-ethylmaleimide (NEM)-sensitive ATPases from the vesicle inside to the outside. ATP addition to cholate-pretreated, but not to intact, vesicles causes H⁺ uptake as visualized by the delta pH indicator, acridine orange. The reoriented H⁺-pump is electrogenic because permeant extravesicular anions or intravesicular K⁺ plus valinomycin enhance H⁺ transport. ATP stimulates H⁺ uptake with an apparent K_m of 93 microM. Support of H⁺ uptake and P_i liberation by ATP greater than GTP approximately ITP greater than UTP indicates a preference for ATP and utilization of other nucleotides at lower efficiency. ADP is a potent, competitive inhibitor of ATP-driven H⁺ uptake (K_i, 24 microM), Mg²⁺ and Mn²⁺ support ATP-driven H⁺ uptake, but Ca²⁺, Ba²⁺, and Zn²⁺ do not, 1 mM Zn²⁺ inhibits MgATP-driven H⁺ transport completely. NEM-sensitive P_i liberation is stimulated by Mg²⁺ and Mg²⁺ and, unlike H⁺ uptake, also by Ca²⁺ suggesting Ca²⁺-dependent ATP hydrolysis unrelated to H⁺ transport. The inside-out oriented H⁺-pump is relatively insensitive toward oligomycin, azide, N,N'-dicyclohexylcarbodiimide (DCCD) and vanadate, but efficiently inhibited by NEM (apparent K_i, 0.77 microM), and 4-chloro-7-nitro-benzoxa-1,3-diazole (NBD-Cl; apparent Ki, 0.39 microM). Taken together, the H⁺-ATPase of proximal tubular brush-border membranes exhibits characteristics very similar to those of "vacuolar type" (V-type) H⁺-ATPases. Hence, V-type H⁺-ATPases occur not only in intracellular organelles but also in specialized plasma membrane areas.