ATP-dependent Na+ transport in cardiac sarcolemmal vesicles

Abstract

Although the enzyme (Na+ + K+)-ATPase has been extensively characterized, few studies of its major role, ATP-dependent Na+ pumping, have been reported in vesicular preparations. This is because it is extremely difficult to determine fluxes of isotopic Na+ accurately in most isolated membrane systems. Using highly purified cardiac sarcolemmal vesicles, we have developed a new technique to detect relative rates of ATP-dependent Na+ transport sensitively. This technique relies on the presence of Na+-Ca2+ exchange and ATP-driven Na+ pump activities on the same inside-out sarcolemmal vesicles. ATP-dependent Na+ uptake is monitored by a subsequent Nai+-dependent Ca2+ uptake reaction (Na+-Ca2+ exchange) using 45Ca2+. We present evidence that the Na+-Ca2+ exchange will be linearly related to the prior active Na+ uptake. Although this method is indirect, it is much more sensitive than a direct approach using Na+ isotopes. Applying this method, we measure cardiac ATP-dependent Na+ transport and (Na+ + K+)-ATPase activities in identical ionic media. We find that the (Na+ + K+)-ATPase and the Na+ pump have identical dependencies on both Na+ and ATP. The dependence on [Na+] is sigmoidal, with a Hill coefficient of 2.8. Na+ pumping is half-maximal at [Na+] = 9 mM. The Km for ATP is 0.21 mM. ADP competitively inhibits ATP-dependent Na+ pumping. This approach should allow other new investigations on ATP-dependent Na+ transport across cardiac sarcolemma.