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

Abstract

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 IC 50s 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 cocentration (5 mM) of K +, suggests that VS may be an inactive isoform of brain Na +, K +-ATPase under resting conditions.