Insulin increases plasma membrane content and reduces phosphorylation of Na+-K+ pump alpha(1)-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 tissue s differs, in part, because of differences in the isoform complement of the catalytic alpha -subunit of the Na+-K+ pump. To analyze specifically the e ffect of insulin on the alpha (1)-isoform of the pump, we have studied huma n embryonic kidney (HEK)-293 cells stably transfected with the rat Na+-K+ p ump alpha (1)-isoform tagged on its first exofacial loop with a hemagglutin in (HA) epitope. The plasma membrane content of alpha (1)-subunits was quan titated by binding a specific HA antibody to intact cells. Insulin rapidly increased the number of alpha (1)-subunits at the cell surface. This gain w as sensitive to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin and to the protein kinase C (PKC) inhibitor bisindolylmaleimide. Furthermo re, the insulin-stimulated gain in surface alpha -subunits correlated with an increase in the binding of an antibody that recognizes only the nonphosp horylated form of alpha (1) (at serine-18). These results suggest that insu lin regulates the Na+-K+ pump in HEK-293 cells, at least in part, by decrea sing serine phosphorylation and increasing plasma membrane content of alpha (1)-subunits via a signaling pathway involving PI 3-kinase and PKC.