The Na(+)-K(+)-ATPase, or sodium pump, is the membrane-bound enzyme that maintains the Na(+) and K(+) gradients across the plasma membrane of animal cells. Because of its importance in many basic and specialized cellular functions, this enzyme must be able to adapt to changing cellular and physiological stimuli. This review presents an overview of the many mechanisms in place to regulate sodium pump activity in a tissue-specific manner. These mechanisms include regulation by substrates, membrane-associated components such as cytoskeletal elements and the gamma-subunit, and circulating endogenous inhibitors as well as a variety of hormones, including corticosteroids, peptide hormones, and catecholamines. In addition, the review considers the effects of a range of specific intracellular signaling pathways involved in the regulation of pump activity and subcellular distribution, with particular consideration given to the effects of protein kinases and phosphatases.

The ␥ subunit of the Na,K-ATPase is a member of the FXYD family of type 2 transmembrane proteins that probably function as regulators of ion transport. Rat ␥ is present primarily in the kidney as two main splice variants, ␥ a and ␥ b , which differ only at their extracellular N termini (TELSANH and MDRWYL, respectively; Kuster, B., Shainskaya, A., Pu, H. X., Goldshleger, R., Blostein, R., Mann, M., and Karlish, S. J. D. (2000) J. Biol. Chem. 275, 18441-18446). Expression in cultured cells indicates that both variants affect catalytic properties, without a detectable difference between ␥ a and ␥ b . At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat ␣1-transfected HeLa cells, i.e. (i) an increase in apparent affinity for ATP, probably secondary to a left shift in E 1 7 E 2 conformational equilibrium and (ii) an increase in K ؉ antagonism of cytoplasmic Na ؉ activation. Antibodies against the C terminus common to both variants (anti-␥) abrogate the first effect but not the second. In contrast, ␥ a and ␥ b show differences in their localization along the kidney tubule. Using anti-␥ (C-terminal) and antibodies to the rat ␣ subunit as well as antibodies to identify cell types, double immunofluorescence showed ␥ in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both ␥ a and ␥ b . In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express ␥ but at lower levels. Antibodies specific for ␥ a and ␥ b showed differences in their cortical location; ␥ a is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, ␥ b but not ␥ a is present in the cortical TAL only. Thus, the importance of ␥ a and ␥ b may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve.A small membrane protein, ␥, first described over 20 years ago in purified kidney Na,K-ATPase preparations (1, 2) associates, in approximately equimolar amounts, with the ␣ and ␤ subunits (3, 4). Molecular cloning of the ␥ subunits of rat, mouse, cow, and sheep indicated a molecular weight of ϳ6500 (5). Cloning and sequencing of the human (6) and Xenopus laevis (7) ␥ subunits have also been reported. Comparison of sequences shows ϳ75% homology among ␥ subunits of the aforementioned different species but is much higher (93%) for only mammalian sequences. Further structural analysis has shown that ␥ comprises a single transmembrane domain and has an N terminus-out, C terminus-in topology (7,8). In addition, two major forms have been recently identified at the molecular level as described below.On SDS-polyacrylamide gel electrophoresis, the ␥ subunit runs as a doublet (apparent molecular masses of ϳ8 and ϳ9 kDa) (5,8), and a doublet is observed following expression in tissue culture cells (8, 9) and in in vitro expression in the pre...

The Na,K-ATPase comprises a catalytic ␣ subunit and a glycosylated ␤ subunit. Another membrane polypeptide, ␥, first described by Forbush et al. (Forbush, B., III, Kaplan, J. H., and Hoffman, J. F. (1978) Biochemistry 17, 3667-3676) associates with ␣ and ␤ in purified kidney enzyme preparations. In this study, we have used a polyclonal anti-␥ antiserum to define the tissue specificity and topology of ␥ and to address the question of whether ␥ has a functional role. The trypsin sensitivity of the amino terminus of the ␥ subunit in intact right-side-out pig kidney microsomes has confirmed that it is a type I membrane protein with an extracellular amino terminus. Western blot analysis shows that ␥ subunit protein is present only in membranes from kidney tubules (rat, dog, pig) and not those from axolemma, heart, red blood cells, kidney glomeruli, cultured glomerular cells, ␣ 1 -transfected HeLa cells, all derived from the same (rat) species, nor from three cultured cell lines derived from tubules of the kidney, namely NRK-52E (rat), LLC-PK (pig), or MDCK (dog). To gain insight into ␥ function, the effects of the anti-␥ serum on the kinetic behavior of rat kidney sodium pumps was examined. The following evidence suggests that ␥ stabilizes E 1 conformation(s) of the enzyme and that anti-␥ counteracts this effect: (i) anti-␥ inhibits Na,K-ATPase, and the inhibition increases at acidic pH under which condition the E 2 (K) 3 E 1 phase of the reaction sequence becomes more ratelimiting, (ii) the oligomycin-stimulated increase in the level of phosphoenzyme was greater in the presence of anti-␥ indicating that the antibody shifts the E 1 7 7 E 2 P equilibria toward E 2 P, and (iii) when the Na ؉ -ATPase reaction is assayed with the Na ؉ concentration reduced to levels (<2 mM) which limit the rate of the E 1 3 3 E 2 P transition, anti-␥ is stimulatory. These observations taken together with evidence that the pig ␥ subunit, which migrates as a doublet on polyacrylamide gels, is sensitive to digestion by trypsin, and that Rb ؉ ions partially protect it against this effect, indicate that the ␥ subunit is a tissue-specific regulator which shifts the steady-state equilibria toward E 1 . Accordingly, binding of anti-␥ disrupts ␣␤-␥ interactions and counteracts these modulatory effects of the ␥ subunit.

The functional role of the ␥ subunit of the Na,KATPase was studied using rat ␥ cDNA-transfected HEK-293 cells and an antiserum (␥C33) specific for ␥. Although the sequence for ␥ was verified and shown to be larger (7237 Da) Overall, our data demonstrate that ␥ is a tissue (kidney)-specific regulator of the Na,K-ATPase that can increase the apparent affinity of the enzyme for ATP in a manner that is reversible by anti-␥ antiserum.The Na,K-ATPase is the sodium pump protein responsible for maintaining the electrochemical gradient present across the membranes of most animal cells (1). It consists of at least two subunits, ␣ and ␤, each of which exists as one of several isoforms

Like the gamma-subunit of Na-K-ATPase, the corticosteroid hormone-induced factor (CHIF) is a member of the FXYD family of one-transmembrane-segment proteins. Both CHIF and two splice variants of gamma, gamma(a) and gamma(b), are expressed in the kidney. Immunolocalization experiments demonstrate mutually exclusive expression of CHIF and gamma in different nephron segments. Specific coimmunoprecipitation experiments demonstrate the existence in kidney membranes of the complexes alpha/beta/gamma(a), alpha/beta/gamma(b), and alpha/beta/CHIF and exclude mixed complexes such as alpha/beta/gamma(a)/gamma(b) and alpha/beta/gamma/CHIF. CHIF has been expressed in HeLa cells harboring the rat alpha(1)-subunit of Na-K-ATPase. (86)Rb flux experiments demonstrate that CHIF induces a two- to threefold increase in apparent affinity for cytoplasmic Na (K'(Na)) but does not affect affinity for extracellular K (Rb) ions (K'(K)) or V(max). Measurements of Na-K-ATPase using isolated membranes show similar but smaller effects of CHIF on K'(Na), whereas K'(K) and K'(ATP) are unaffected. The functional effects of CHIF differ from those of gamma. An implication of these findings is that other FXYD proteins could act as tissue-specific modulators of Na-K-ATPase.

The alpha2 isoform of the Na,K-ATPase exhibits kinetic behavior distinct from that of the alpha1 isoform. The distinctive behavior is apparent when the reaction is carried out under conditions (micromolar ATP concentration) in which the K+ deocclusion pathway of the reaction cycle is rate-limiting; the alpha1 activity is inhibited by K+, whereas alpha2 is stimulated. When 32 NH2-terminal amino acid residues are removed from alpha1, the kinetic behavior of the mutant enzyme (alpha1M32) is similar to that of alpha2 (Daly, S. E., Lane, L. K., and Blostein, R. (1994) J. Biol. Chem. 269, 23944-23948). In the current study, the region of the alpha1 NH2 terminus involved in modulating this kinetic behavior has been localized to the highly charged sequence comprising residues 24-32. Within this nonapeptide, differences between alpha1 and alpha2 are conservative and are confined to residues 25-27. The behavior of two chimeric enzymes: (i) alpha1 with the first 32 residues identical to the alpha2 sequence, alpha1 (1-32alpha2), and (ii) alpha2 with the first 32 residues identical to the alpha1 sequence, alpha2(1-32alpha1), indicates that the distinctive kinetic behavior of alpha1 and alpha2 is not due to the 24-32 NH2-terminal domain, per se, but rather to its interaction with other, isoform-specific region(s) of the alpha1 protein. We also demonstrate that the distinct K+ activation profiles of either alpha2 or alpha1M32, compared to alpha1 is due to a faster release of K+ from the K+-occluded enzyme, and to a higher affinity for ATP. This was determined in studies using two approaches: (i) kinetic analysis of the reaction modeled according to a branched pathway of K+ deocclusion through low and high affinity ATP pathways and, (ii) measurements of the (rapid) phosphorylation of the enzyme (E1 conformation) by [gamma-32P]ATP following the rate-limiting formation of the K+-free enzyme from the K+-occluded state (E2(K) --> E1 + K+). The observed kinetic differences between alpha2 and alpha1 suggest that these Na,K-ATPase isoforms differ in the steady-state distribution of E1 and E2 conformational states.

The experiments described in this report reconcile some of the apparent differences in isoform-specific kinetics of the Na,K-ATPase reported in earlier studies. Thus, tissue-specific differences in Na+ and K+ activation kinetics of Na,K-ATPase activity of the same species (rat) were observed when the same isoform was assayed in different tissues or cells. In the case of alpha1, alpha1-transfected HeLa cell, rat kidney, and axolemma membranes were compared. For alpha3, the ouabain-insensitive alpha3*-transfected HeLa cell (cf. Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925-16930), pineal gland, and axolemma (mainly alpha3) membranes were compared. The order of apparent affinities for Na+ of alpha1 pumps was axolemma approximately rat alpha1-transfected HeLa > kidney, and for K+, kidney approximately alpha1-transfected HeLa > axolemma. For alpha3, the order of apparent affinities for Na+ was pineal gland approximately axolemma > alpha3*-transfected HeLa, and for K+, alpha3*-transfected HeLa > axolemma approximately pineal gland. In addition, the differences in apparent affinities for Na+ of either kidney alpha1 or HeLa alpha3* as compared to the same isoform in other tissues were even greater when the K+ concentration was increased. A kinetic analysis of the apparent affinities for Na+ as a function of K+ concentration indicates that isoform-specific as well as tissue-specific differences are related to the apparent affinities for both Na+ and K+, the latter acting as a competitive inhibitor at cytoplasmic Na+ activation sites. Although the nature of the tissue-specific modulation of K+/Na+ antagonism remains unknown, an analysis of the nature of the beta isoform associated with alpha1 or alpha3 using isoform-specific immunoprecipitation indicates that the presence of distinct beta subunits does not account for differences of alpha1 of kidney, axolemma, and HeLa, and of alpha3 of axolemma and HeLa; in both instances beta1 is the predominant beta isoform present or associated with either alpha1 or alpha3. However, a kinetic difference in K+/Na+ antagonism due to distinct betas may apply to alpha3 of axolemma (alpha3beta1) and pineal gland ( alpha3beta2).

The two variants of the ␥ subunit of the rat renal sodium pump, ␥ a and ␥ b , have similar effects on the Na,K-ATPase. Both increase the affinity for ATP due to a shift in the enzyme's E 1 7 E 2 conformational equilibrium toward E 1 . In addition, both increase K ؉ antagonism of cytoplasmic Na ؉ activation. To gain insight into the structural basis for these distinct effects, extramembranous N-terminal and C-terminal mutants of ␥ were expressed in rat ␣1-transfected HeLa cells. At the N terminus, the variant-distinct region was deleted (␥N⌬7) or replaced by alanine residues (␥N7A). At the C terminus, four (␥ a C⌬4) or ten (␥ a C⌬10) residues were deleted. None of these mutations abrogates the K ؉ /Na ؉ antagonism as evidenced in a similar increase in K Na seen at high (100 mM) K ؉ concentration. In contrast, the C-terminal as well as N-terminal deletions (␥N⌬7, ␥ a C⌬4, and