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.

Renal Na(+)-K(+)-ATPase is associated with the gamma-subunit (FXYD2), a single-span membrane protein that modifies ATPase properties. There are two splice variants with different amino termini, gamma(a) and gamma(b). Both were found in the inner stripe of the outer medulla in the thick ascending limb. Coimmunoprecipitation with each other and the alpha-subunit indicated that they were associated in macromolecular complexes. Association was controlled by ligands that affect Na(+)-K(+)-ATPase conformation. In the cortex, the proportion of the gamma(b)-subunit was markedly lower, and the gamma(a)-subunit predominated in isolated proximal tubule cells. By immunofluorescence, the gamma(b)-subunit was detected in the superficial cortex only in the distal convoluted tubule and connecting tubule, which are rich in Na(+)-K(+)-ATPase but comprise a minor fraction of cortex mass. In the outer stripe of the outer medulla and for a short distance in the deep cortex, the thick ascending limb predominantly expressed the gamma(b)-subunit. Because different mechanisms maintain and regulate Na(+) homeostasis in different nephron segments, the splice forms of the gamma-subunit may have evolved to control the renal Na(+) pump through pump properties, gene expression, or both.

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.

Thermal denaturation can help elucidate protein domain substructure. We previously showed that the Na,K-ATPase partially unfolded when heated to 55°C (Arystarkhova, E., Gibbons, D. L., and Sweadner, K. J. (1995) J. Biol. Chem. 270, 8785-8796). The ␤ subunit unfolded without leaving the membrane, but three transmembrane spans (M8-M10) and the C terminus of the ␣ subunit were extruded, while the rest of ␣ retained its normal topology with respect to the lipid bilayer. Here we investigated thermal denaturation further, with several salient results. First, trypsin sensitivity at both surfaces of ␣ was increased, but not sensitivity to V8 protease, suggesting that the cytoplasmic domains and extruded domain were less tightly packed but still retained secondary structure. Second, thermal denaturation was accompanied by SDS-resistant aggregation of ␣ subunits as dimers, trimers, and tetramers without ␤ or ␥ subunits. This implies specific ␣-␣ contact. Third, the ␥ subunit, like the C-terminal spans of ␣, was selectively lost from the membrane. This suggests its association with M8-M10 rather than the more firmly anchored transmembrane spans. The picture that emerges is of a Na,K-ATPase complex of ␣, ␤, and ␥ subunits in which ␣ can associate in assemblies as large as tetramers via its cytoplasmic domain, while ␤ and ␥ subunits associate with ␣ primarily in its C-terminal portion, which has a unique structure and thermal instability.

The Na,K-ATPase is a dominant factor in retinal energy metabolism, and unique combinations of isoforms of its alpha and beta subunits are expressed in different cell types and determine its functional properties. We used isoform-specific antibodies and fluorescence confocal microscopy to determine the expression of Na,K-ATPase alpha and beta subunits in the mouse and rat retina. In the adult retina, alpha1 was found in Müller and horizontal cells, alpha2 in some Müller glia, and alpha3 in photoreceptors and all retinal neurons. beta1 was largely restricted to horizontal, amacrine, and ganglion cells; beta2 was largely restricted to photoreceptors, bipolar cells, and Müller glia; and beta3 was largely restricted to photoreceptors. Photoreceptor inner segments have the highest concentration of Na,K-ATPase in adult retinas. Isoform distribution exhibited marked changes during postnatal development. alpha3 and beta2 were in undifferentiated photoreceptor somas at birth but only later were targeted to inner segments and synaptic terminals. beta3, in contrast, was expressed late in photoreceptor differentiation and was immediately targeted to inner segments. A high level of beta1 expression in horizontal cells preceded migration, whereas increases in beta2 expression in bipolar cells occurred very late, coinciding with synaptogenesis in the inner plexiform layer. Most of the spatial specification of Na,K-ATPase isoform expression was completed before eye opening and the onset of electroretinographic responses on postnatal day 13 (P13), but quantitative increase continued until P22 in parallel with synaptogenesis.

The ␥ subunit of the Na,K-ATPase, a 7-kDa single-span membrane protein, is a member of the FXYD gene family. Several FXYD proteins have been shown to bind to Na,K-ATPase and modulate its properties, and each FXYD protein appears to alter enzyme kinetics differently. Different results have sometimes been obtained with different experimental systems, however. To test for effects of ␥ in a native tissue environment, mice lacking a functional ␥ subunit gene (Fxyd2) were generated. These mice were viable and without observable pathology. Prior work in the mouse embryo showed that ␥ is expressed at the blastocyst stage. However, there was no delay in blastocele formation, and the expected Mendelian ratios of offspring were obtained even with Fxyd2 ؊/؊ dams. In adult Fxyd2 ؊/؊ mouse kidney, splice variants of ␥ that have different nephron segment-specific expression patterns were absent. Purified ␥-deficient renal Na,K-ATPase displayed higher apparent affinity for Na ؉ without significant change in apparent affinity for K ؉ . Affinity for ATP, which was expected to be decreased, was instead slightly increased. The results suggest that regulation of Na ؉ sensitivity is a major functional role for this protein, whereas regulation of ATP affinity may be context-specific. Most importantly, this implies that ␥ and other FXYD proteins have their effects by local and not global conformation change. Na,K-ATPase lacking the ␥ subunit had increased thermal lability. Combined with other evidence that ␥ participates in an early step of thermal denaturation, this indicates that FXYD proteins may play an important structural role in the enzyme complex.

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In kidney, the Na,K-ATPase is associated with a single span protein, the ␥ subunit (FXYD2). Two splice variants are differentially expressed along the nephron and have a differential influence on Na,K-ATPase when stably expressed in mammalian cells in culture. Here we used a combination of gene induction and gene silencing techniques to test the functional impact of ␥ by means other than transfection. NRK-52E cells (of proximal tubule origin) do not express ␥ as a protein under regular tissue culture conditions. However, when they were exposed to hyperosmotic medium, induction of only the ␥a splice variant was observed, which was accompanied by a reduction in the rate of cell division. Kinetic analysis of stable enzyme properties from control (␣1␤1) and hypertonicity-treated cultures (␣1␤1␥a) revealed a significant reduction (up to 60%) of Na,K-ATPase activity measured under V max conditions with little or no change in the amounts of ␣1␤1. This effect as well as the reduction in cell growth rate was practically abolished when ␥ expression was knocked down using specific small interfering RNA duplexes. Surprisingly, a similar induction of endogenous ␥a because of hypertonicity was seen in rat cell lines of other than renal origin: C6 (glioma), PC12 (pheochromocytoma), and L6 (myoblasts). Furthermore, exposure of NRK-52E cells to other stress inducers such as heat shock, exogenous oxidation, and chemical stress also resulted in a selective induction of ␥a. Taken together, the data imply that induction of ␥a may have adaptive value by being a part of a general cellular response to genotoxic stress.