Differential Regulation of Renal Na,K-ATPase by Splice Variants of the Subunit*

Sodium and potassium-exchanging adenosine triphosphatase (Na,K-ATPase) in the kidney is associated with the subunit ( , FXYD2), a single-span membrane protein that modulates ATPase properties. Rat and human occur in two splice variants, a and b, with different N termini. Here we investigated their structural heterogeneity and functional effects on Na,K-ATPase properties. Both forms were post-translationally modified during in vitro translation with microsomes, indicating that there are four possible forms of . Site-directed mutagenesis revealed Thr and Ser as potential sites for post-translational modification. Similar modification can occur in cells, with consequences for Na,K-ATPase properties. We showed previously that stable transfection of a into NRK-52E cells resulted in reduction of apparent affinities for Na and K . Individual clones differed in post-translational modification, however, and the effect on Na affinity was absent in clones with full modification. Here, transfection of b also resulted in clones with or without post-translational modification. Both groups showed a reduction in Na affinity, but modification was required for the effect on K affinity. There were minor increases in ATP affinity. The physiological importance of the reduction in Na affinity was shown by the slower growth of a, b, and b transfectants in culture. The differential influence of the four structural variants of on affinities of the Na,KATPase for Na and K , together with our previous finding of different distributions of a and b along the rat nephron, suggests a highly specific mode of regulation of sodium pump properties in kidney.