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pubmed:abstractText |
Inside-out membrane vesicles were prepared from human red blood cells pretreated with diisothiocyano-2,2'-disulfonic stilbene to inhibit anion fluxes. The pH-sensitive probe fluorescein isothiocyanate-dextran was incorporated inside the vesicles. Formation of pH gradients due to proton transport by the sodium pump was distinguished from pH gradients formed in response to transmembrane electrical potentials generated by the pump by virtue of their insensitivity and sensitivity, respectively, to dissipation by lipophilic cations. Under the conditions used (pH 6.6), proton transport by the Na,K-ATPase was minimized, and the formation of pH gradients in response to electrical potentials was detected. Thus, the generation of a strophanthidin-sensitive, ATP-dependent electrical potential, inside positive (approximately 1 mV) upon addition of 4 meq of sodium to potassium-filled inside-out vesicles is consistent with the well documented stoichiometry of three sodium ions exchanging with two potassium ions. In contrast, when the cytoplasmic sodium concentration is reduced to less than or equal to 0.4 mM, the potential generated is of the opposite sign, i.e. inside negative, consistent with the decreased Na:K coupling ratio reported previously, i.e. Na:K(Rb) coupling ratios of approximating 1:2 when the sodium concentration is reduced to 0.2 mM (Blostein, R. (1983) J. Biol. Chem. 258, 12228-12232).
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