An electrogenic Na+/Ca2+ antiporter in addition to the Ca2+ pump in cardiac sarcolemma

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na+ + K+)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca2+ transport. The Ca2+ pump was stimulated 1.7- and 2.1-fold by external Na+ and K+, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca2+ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca2+ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca2+ in a medium containing 160 mM NaCl. Ca2+ movements were also studied in the absence of ATP and Mg2+. Vesicles containing an outwardly directed Na+ gradient showed the highest Ca2+ uptake activity. These findings suggested the operation of a Ca2+/Na+ antiporter in addition to the active Ca2+ pump in these sarcolemmal vesicles. A valinomycin-induced inward K+-diffusion potential stimulated the Na+-Ca2+ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg2+ the transmembrane Nai+/Nao+ gradient exceeded 160/15 mM concentrations, Ca2+ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na+ gradient-induced Ca2+ uptake to be a translocation of Ca2+ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na+-Ca2+ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.