Insulin increases plasma membrane content and reduces phosphorylation of Na⁺-K⁺ pump α₁-subunit in HEK-293 cells

Insulin stimulates K⁺ uptake and Na⁺ efflux via the Na⁺-K⁺ pump in kidney, skeletal muscle, and brain. The mechanism of insulin action in these tissues differs, in part, because of differences in the isoform complement of the catalytic α-subunit of the Na⁺-K⁺ pump. To analyze specifically the effect of insulin on the α₁-isoform of the pump, we have studied human embryonic kidney (HEK)-293 cells stably transfected with the rat Na⁺-K⁺ pump α₁-isoform tagged on its first exofacial loop with a hemagglutinin (HA) epitope. The plasma membrane content of α₁-subunits was quantitated by binding a specific HA antibody to intact cells. Insulin rapidly increased the number of α₁-subunits at the cell surface. This gain was sensitive to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin and to the protein kinase C (PKC) inhibitor bisindolylmaleimide. Furthermore, the insulin-stimulated gain in surface α₁-subunits correlated with an increase in the binding of an antibody that recognizes only the nonphosphorylated form of α₁ (at serine-18). These results suggest that insulin regulates the Na⁺-K⁺ pump in HEK-293 cells, at least in part, by decreasing serine phosphorylation and increasing plasma membrane content of α₁-subunits via a signaling pathway involving PI 3-kinase and PKC.