Dopamine inhibits Na؉ ,K ؉ -ATPase activity in renal tubule cells. This inhibition is associated with phosphorylation and internalization of the ␣ subunit, both events being protein kinase C-dependent. Studies of purified preparations, fusion proteins with site-directed mutagenesis, and heterologous expression systems have identified two major protein kinase C phosphorylation residues (Ser-11 and Ser-18) in the rat ␣ 1 subunit isoform. To identify the phosphorylation site(s) that mediates endocytosis of the subunit in response to dopamine, we have performed site-directed mutagenesis of these residues in the rat ␣ 1 subunit and expressed the mutated forms in a renal epithelial cell line. Dopamine inhibited Na ؉ ,K ؉ -ATPase activity and increased ␣ subunit phosphorylation and clathrin-dependent endocytosis into endosomes in cells expressing the wild type ␣ 1 subunit or the S11A ␣ 1 mutant, and both effects were blocked by protein kinase C inhibition. In contrast, dopamine did not elicit any of these effects in cells expressing the S18A ␣ 1 mutant. While Ser-18 phosphorylation is necessary for endocytosis, it does not affect per se the enzymatic activity: preventing endocytosis with wortmannin or LY294009 blocked the inhibitory effect of dopamine on Na ؉ ,K ؉ -ATPase activity, although it did not alter the increased ␣ subunit phosphorylation induced by this agonist.We conclude that dopamine-induced inhibition of Na ؉ ,K ؉ -ATPase activity in rat renal tubule cells requires endocytosis of the ␣ subunit into defined intracellular compartments and that phosphorylation of Ser-18 is essential for this process.

Abstract-␣-Adducin polymorphism in humans is associated with abnormal renal sodium handling and high blood pressure. The mechanisms by which mutations in adducin affect the renal set point for sodium excretion are not known. Decreases in Na ϩ ,K ϩ -ATPase activity attributable to endocytosis of active units in renal tubule cells by dopamine regulates sodium excretion during high-salt diet. Milan rats carrying the hypertensive adducin phenotype have a higher renal tubule Na ϩ ,K ϩ -ATPase activity, and their Na ϩ ,K ϩ -ATPase molecules do not undergo endocytosis in response to dopamine as do those of the normotensive strain. Dopamine fails to promote the interaction between adaptins and the Na ϩ ,K ϩ -ATPase because of adaptin-2 subunit hyperphosphorylation. Expression of the hypertensive rat or human variant of adducin into normal renal epithelial cells recreates the hypertensive phenotype with higher Na ϩ ,K ϩ -ATPase activity, 2-subunit hyperphosphorylation, and impaired Na ϩ ,K ϩ -ATPase endocytosis. Thus, increased renal Na ϩ ,K ϩ -ATPase activity and altered sodium reabsorption in certain forms of hypertension could be attributed to a mutant form of adducin that impairs the dynamic regulation of renal Na ϩ ,K ϩ -ATPase endocytosis in response to natriuretic signals. Key Words: high blood pressure Ⅲ sodium retention Ⅲ protein trafficking Ⅲ cytoskeleton Ⅲ endocytosis S pontaneous mutations in ␣-adducin gene favor renal sodium retention and the development of high blood pressure in humans and rodents. 1 The estimated frequency of the mutated allele fluctuates among 8% in African Americans, 15% to 20% in whites, and 50% in Japanese individuals. The resulting phenotype is similar in rats and humans despite species differences in the mutation sites (human G460W/ S586C; rats F316Y). These polymorphisms affect the pressure natriuresis curve, the lithium clearance (an indicator of proximal tubule [PCT] sodium transport), 2 and the chronic blood pressure response to diuretics. 3,4 Recent studies further substantiate the role of adducin in human hypertension and the associated renal or cardiac alterations. 5,6 Many of these findings have been obtained in low-renin hypertensive patients, 7 in whom renal Na ϩ retention is a particularly important pathogenetic factor, or in patients carrying the gene variants known to affect renal Na ϩ handling. 8 At the cellular level, adducin plays an active role in cytoskeleton organization by regulating actin dynamics. 9 The mutated hypertensive variant of the adducin ␣-subunit increases the rate of actin polymerization and bundling in a cell-free system compared with the wild-type variant. 10 In cultured renal cells stably transfected with the mutated ␣-adducin, the actin cytoskeleton network appears thicker and Na ϩ ,K ϩ -ATPase activity is higher than in cells transfected with the wild-type variant. 10 In renal epithelial cells, the Na ϩ ,K ϩ -ATPase is located at the basolateral membrane domain and is critical for vectorial sodium transport. Its regulation by catecholamines cont...