Electrogenic and electroneutral transport modes of renal Na/K ATPase reconstituted into proteoliposomes

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Abstract
This paper describes measurements of electrical potentials generated by renal Na/K-ATPase reconstituted into proteoliposomes, utilizing the anionic dye, oxonol VI. Calibration of absorption changes with imposed diffusion potentials allows estimation of absolute values of electrogenic potentials. ATP-dependent Nacyt/Kexc exchange in K-loaded vesicles generates large potentials, up to 250 mV. By comparing initial rates or steady-state potentials with ATP-dependent22Na fluxes in different conditions, it is possible to infer whether coupling ratios are constant or variable. For concentrations of Nacyt (2–50mm) and ATP (1–1000 μm) and pH's (6.5–8.5), the classical 3Nacyt/2Kexc coupling ratio is maintained. However, at low Nacyt concentrations (cyt/2Kexc. ATP-dependent Nacyt/congenerexc exchange in vesicles loaded with Rb, Cs, Li and Na is electrogenic. In this mode congeners, including Naexc, act as Kexc surrogates in an electrogenic 3Nacyt/2congenerexc exchange. (ATP+Pi)-dependent Kcyt/Kexc exchange in K-loaded vesicles is electroneutral. ATP-dependent “uncoupled” Na flux into Na- and K-free vesicles is electroneutral at pH 6.5–7.0 but becomes progressively electrogenic as the pH is raised to 8.5. The22Na flux shows no anion specificity. We propose that “uncoupled” Na flux is an electroneutral 3Nacyt/3Hexc exchange at pH 6.5–7.0 but at higher pH's the coupling ratio changes progressively, reaching 3Na/no ions at pH 8.5. Slow passive pump-mediated net K uptake into Na- and K-free vesicles is electroneutral, and may also involve Kcyt/Hexc exchange. We propose the general hypothesis that coupling ratios are fixed when cation transport sites are saturated, but at low concentrations of transported cations, e.g., Nacyt in Na/K exchange and Hexc in “uncoupled” Na flux, coupling ratios may change.