Characterization of an electrogenic ATP and chloride-dependent proton translocating pump from rat renal medulla.

J D Kaunitz,R D Gunther,G Sachs

doi:10.1016/s0021-9258(17)39069-5

Abstract

To study acidification mechanisms in the distal nephron, microsomes were prepared from rat renal medulla by differential centrifugation. Microsomes were enriched in the enzyme marker gamma-glutamyl transferase and contained an ATP-dependent proton pump, as evidenced by ATP-dependent, 3,3',4',5-tetrachlorosalicylanilide-reversible quenching of acridine orange fluorescence. Acidification was vanadate-insensitive, but was completely inhibited by micromolar N-ethylmaleimide. Maximal acidification was achieved in the presence of halide (Cl-, Br-) only and was not attainable with potassium-valinomycin diffusion potentials without halide ion. Microsomal ATPase activity was neither chloride- nor N-ethylmaleimide-sensitive. A chloride conductance was observed only with vesicles which had undergone ATP-dependent acidification. An ATP-dependent, N-ethylmaleimide-inhibitable, 3,3',4',5-tetrachlorosalicylanilide-reversible, and chloride-attenuated quench of bis(1,3-dibutylbarbituric acid-(5] pentamethinoxonol fluorescence was seen, consistent with net transfer of positive charge into the vesicles. Nonetheless, positive intravesicular potentials increased the ATP-dependent initial acidification rate, perhaps by increasing availability of chloride ion to the transport site. Our results are consistent with an electrogenic, ATP-dependent proton pump regulated by a voltage-sensitive chloride site.

Highlights

IntroductionMale Wistar rats (150-300g) were was completely inhibited by micromolar N-ethylmaleimide.Maximalacidification was achievedinthe presence of halide (Cl-, Br-) only and was not attaindecapitated and exsanguinated
We found ATP-dependent proton uptake into microsomes from renal medulla with inhibitor sensitivities and ion requirements similar to other cation-nonspecific vanadate-insensitive proton pumps, and different from mitochondria
Studies of the' effect of anion gradients and membrane potentials (E,) on pump rate are consistent with an electrogenic H+ pump whose rate is increased by halide ion and positive intravesicular potentials

Summary

Introduction

Male Wistar rats (150-300g) were was completely inhibited by micromolar N-ethylmaleimide.Maximalacidification was achievedinthe presence of halide (Cl-, Br-) only and was not attaindecapitated and exsanguinated. All operations and solutions after removal of the medullae were at 2 “C. The kidneys were rapidly removed and placed in anice-coldisotonic buffer of lowionic strength (250 mM sucrose, 20 mM PIPES’/Tris, pH 7.4). The perinephric fat able with potassium-valinomycin diffusion potentials was removed, and the kidneys were lon~tudinallydivided with a without halide ion. Positive intravesicular potentials increased the ATP-dependentinitial acidification rate, perhapsbyincreasing ayailability of chloride ion to the transposrite. The approximate yield of the above procedure was 1mg of membrane protein/rat. E q m e Assays dependent protonpump regulated by avoltage-sensi- ATPase-A modification of a previously published method [7]was tive chloride site.

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Results

Conclusion