Elucidation of mechanisms regulating Na pump isoform activities after neuronal excitation.

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We investigated expression and regulation of Na pump isoforms in cultured cerebellar granule cells. The cells expressed three Na pump isoforms (α1, α2 and α3 isoforms), however the α1 isoform acted as a main ion pump under basal conditions. The ion pump activity of the α2/α3 isoforms increased remarkably after stimulation of the neurons with glutamate as reported previously in cultured cerebral neurons. The glutamate effects were mainly mediated by non-NMDA receptors. We examined mechanism of the differential regulation of the isoform activities in cultured cerebral neurons. An efficiency of K^+ transport of the α2/α3 isoform was lower than that of the α1 isoform under basal conditions. The low efficiency was due to inhibition of the α2/α3 isoform by physiological concentrations of extracellular potassium. The α2/α3 isoform activity was remarkably inhibited at more than 1 mM K^+, but not the a1 isoform. In contrast, the inhibition of the α2/α3 isoform by potassium was vanished after glutamate excitation of the neurons. Incubating the neurons with KN-93 (inhibitor of CaM kinase II) or W-7 (calmodulin antagonist) released the α2/α3 isoform from the inhibition by extracellular potassium. The transport efficiency of the α2/α3 isoform was raised without increasing the total K^+ uptake activity. Stimulation of the neurons with monensin (sodium ionophore) in the presence of KN-93 increased the total activity and mimicked the effects of glutamate excitation. These results suggest that glutamate excitation of the neurons increases the α2/α3 isoform activity by two mechanisms. First it releases the α2/α3 isoform from the inhibition by extracellular potassium, and second it activated the isoform by increasing intracellular sodium concentration. We also found α subunit of the Na pump was phosphorylated in situ under basal conditions, and an extent of the phosphorylation of the α subunit changed after stimulation the neurons with glutamate agonists.