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Electrogenicity of Ca2+ transport catalyzed by the Ca2+-ATPase from sarcoplasmic reticulum.

The Ca’+-ATPase was isolated from sarcoplasmic reticulum and reconstituted into liposomes by the freezethaw-sonication procedure of Kasahara and Hinkle (Kasahara, M., and Hinkle, P. C. (1977) J. Biol. Chem 252, 7384-7390) with egg yolk phosphatidylcholine as the only phospholipid. By varying the ion concentrations during reconstitution and assay, different K+ gradients across the membrane were established, permitting the creation of controlled membrane potentials in the presence of valinomycin. Stimulation or inhibition of the rate of ATP-dependent Ca2+ uptake was found to be a function of the electrical potential difference imposed on the vesicular membrane. The potential difference which did not affect the rate of Ca2+ transport, + 61 + 10 mV (positive inside the vesicles), was considered to reflect the membrane potential established by an electrogenic, enzyme-mediated Ca2’ uptake into the vesicles. The membrane potential developing in the reconstituted vesicles during active Ca2+ pumping was measured independently with a fluorescent probe, %anilino-1-naphthalenesulfonic acid. The value of +51 mV was obtained, in good agreement with the first measurement. The electrogenicity of Ca2’ transport in a reconstituted system was further confirmed by experiments in which an electrical potential difference across the vesicle membrane was induced by addition of lipophilic anions. The relevance of these findings to the mechanism of ion transport in native sarcoplasmic reticulum is discussed.