Identification of a third isoform of Na+, K(+)-ATPase activity in rat brain synaptosomes

3H-ouabain binding and ouabain-inhibitable 86Rb+ (K+) uptake were investigated as a means to identify a third isoform of Na+, K(+)-ATPase in crude synaptosome preparations. The specific binding of low concentrations (10 nM and 1 uM) of 3H-ouabain, in crude synaptosome preparations, was markedly inhibited by K+ (0.5-5 mM). Accordingly, 86Rb+ (K+) uptake, in the presence of 5 mM K+ was not sensitive to inhibition by low concentrations (10(-11)-10(-7) M) of ouabain. Higher concentrations (10(-6)-10(-2.6) M) of ouabain resulted in a biphasic inhibition of K+ uptake, which distinguished the activities of the presumed alpha 2 and alpha 1 isozymes of Na+, K(+)-ATPase. Reduction of K+ (1.25 mM and 0.5 mM) in the incubation, resulted in the observation of a third component of ouabain-sensitive K+ uptake. This Na+, K(+)-ATPase activity, which was defined, pharmacologically, as very sensitive (VS) to ouabain, exhibited IC50S of 3.6 nM and 92 nM at 1.25 mM K+ and 0.5 mM K+, respectively. Inhibition of ouabain binding and VS-dependent K+ uptake, at a high, physiological concentration (5 mM) of K+, suggests that VS may be an inactive isoform of brain Na+, K(+)-ATPase under resting conditions.