The Na؉ ,K ؉ -ATPase catalyzes the active transport of ions. It has two necessary subunits, ␣ and ␤, but in kidney it is also associated with a 7.4-kDa protein, the ␥ subunit. Stable transfection was used to determine the effect of ␥ on Na,K-ATPase properties. When isolated from either kidney or transfected cells, ␣␤␥ had lower affinities for both Na ؉ and K ؉ than ␣␤. A post-translational modification of ␥ selectively eliminated the effect on Na ؉ affinity, suggesting three configurations (␣␤, ␣␤␥, and ␣␤␥*) conferring different stable properties to Na,K-ATPase. In the nephron, segment-specific differences in Na ؉ affinity have been reported that cannot be explained by the known ␣ and ␤ subunit isoforms of Na,K-ATPase. Immunofluorescence was used to detect ␥ in rat renal cortex. Cortical ascending limb and some cortical collecting tubules lacked ␥, correlating with higher Na ؉ affinities in those segments reported in the literature. Selective expression in different segments of the nephron is consistent with a modulatory role for the ␥ subunit in renal physiology.

Phospholemman (FXYD1) is a homolog of the Na,K-ATPase gamma subunit (FXYD2), a small accessory protein that modulates ATPase activity. Here we show that phospholemman is highly expressed in selected structures in the CNS. It is most abundant in cerebellum, where it was detected in the molecular layer, in Purkinje neurons, and in axons traversing the granule cell layer. Phospholemman was particularly enriched in choroid plexus, the organ that secretes CSF in the ventricles, where it colocalized with Na,K-ATPase in the apical membrane. It was also enriched, with Na,K-ATPase, in certain tanycytes or ependymal cells of the ventricle wall. Two different experimental approaches demonstrated that phospholemman physically associated with the Na,K-ATPase in cerebellum and choroid plexus: the proteins copurified after detergent treatment and co-immunoprecipitated from solubilized crude membranes using either anti-phospholemman or anti-Na,K-ATPase antibodies. Phospholemman antibodies precipitated all three Na,K-ATPase alpha subunit isoforms (alpha1-alpha3) from cerebellum, indicating that the interaction is not specific to a particular alpha isoform and consistent with the presence of phospholemman in both neurons and glia. Antibodies against the C-terminal domain of phospholemman reduced Na,K-ATPase activity in vitro without effect on Na+ affinity. At least two other FXYD family members have been detected in the CNS, suggesting that additional complexity of sodium pump regulation will be found.

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 2 and Ser 5 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.