There are four isoforms of the ␣ subunit (␣1-4) and three isoforms of the ␤ subunit (␤1-3) of Na,K-ATPase, with distinct tissue-specific distribution and physiological functions. ␣2 is thought to play a key role in cardiac and smooth muscle contraction and be an important target of cardiac glycosides. An ␣2-selective cardiac glycoside could provide important insights into physiological and pharmacological properties of ␣2. The isoform selectivity of a large number of cardiac glycosides has been assessed utilizing ␣1␤1, ␣2␤1, and ␣3␤1 isoforms of human Na,K-ATPase expressed in Pichia pastoris and the purified detergent-soluble isoform proteins. Binding affinities of the digitalis glycosides, digoxin, ␤-methyl digoxin, and digitoxin show moderate but highly significant selectivity (up to 4-fold) for ␣2/␣3 over ␣1 (K D ␣1 > ␣2 ‫؍‬ ␣3). By contrast, ouabain shows moderate selectivity (≈2.5-fold) for ␣1 over ␣2 (K D ␣1 < ␣3 < ␣2). Binding affinities for the three isoforms of digoxigenin, digitoxigenin, and all other aglycones tested are indistinguishable (K D ␣1 ‫؍‬ ␣3 ‫؍‬ ␣2), showing that the sugar determines isoform selectivity. Selectivity patterns for inhibition of Na,K-ATPase activity of the purified isoform proteins are consistent with binding selectivities, modified somewhat by different affinities of K ؉ ions for antagonizing cardiac glycoside binding on the three isoforms. The mechanistic insight on the role of the sugars is strongly supported by a recent structure of Na,K-ATPase with bound ouabain, which implies that aglycones of cardiac glycosides cannot discriminate between isoforms. In conclusion, several digitalis glycosides, but not ouabain, are moderately ␣2-selective. This supports a major role of ␣2 in cardiac contraction and cardiotonic effects of digitalis glycosides.

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.

Tryptic digestion of pig renal Na/K-ATPase in the presence ofRb and absence of Ca ions removes about half of the protein but leaves a stable 19-kDa membrane-embedded fragment derived from the et chain, a largely intact ( chain, and essentially normal Rb-and Na-occlusion capacity. Subsequent digestion with trypsin in the presence of Ca or absence of Rb ions leads to rapid loss of the 19-kDa fragment and a parallel loss of Rb occlusion, demonstrating that the fragment is essential for occlusion. The N-terminal sequence of the 19-kDa fragment is Asn-Pro-Lys-Thr-Asp-Lys-Leu-ValAsn-Glu-Arg-Leu-Ile-Ser-Met-Ala, beginning at residue 830 and extending toward the C terminus. Membranes containing the 19-kDa fragment have the following functional properties. (i) ATP-dependent functions are absent. (ii) The apparent affinity for occluding Rb is unchanged, the affinity for Na is lower than in the control enzyme, and activation is now strongly sigmoidal rather than hyperbolic. (iii) Membranes containing the 19-kDa fragment can be reconstituted into phospholipid vesicles and sustain slow Rb-Rb exchange. Thus the transport pathway is retained. We conclude that cation occlusion sites and the transport pathway within transmembrane segments are quite separate from the ATP binding site, located on the cytoplasmic domain of the a chain. Interactions between cation and ATP sites, the heart of active transport, must be indirect-mediated, presumably, by conformational changes of the protein.The Na/K-ATPase or Na/K-pump has been purified, cloned, and sequenced (1,2). Much is known about transport modes, ion occlusion, and conformational changes (3). Nevertheless, the central question-how the free energy of hydrolysis of ATP is transduced into active pumping of cations-remains unanswered. ATP-binding residues are located on the central cytoplasmic loop of the a chain (1, 2), but little is known about the cation binding sites or pathway for cation movement (for a review, see ref. 4). Information on the cation binding sites would undoubtedly clarify the mechanism of coupling. In particular, by knowing whether the cation and ATP sites were adjacent or quite separate, one could distinguish between more or less direct mechanisms.One approach to identifying cation-binding residues is chemical labeling. Carboxyl residues are good candidates, and hence reagents such as N,N'-dicyclohexylcarbodiimide (DCCD) have been used extensively (5-8). We (8, 9) have studied inactivation by DCCD of Rb and Na occlusion and concluded that carboxyl residues are involved and Rb and Na bind to the same residues. Incorporation of about 2 mol of DCCD per mol of a chain accompanies full inactivation of Rb occlusion (10). Subsequent attempts to locate the label in the primary sequence could exploit selective proteolysis (11). In control experiments designed to produce extensive digestion, we were surprised to find a condition in which occlusion was retained while the a chain was largely but incompletely fragmented. In this condition a stable 19-kDa membraneemb...

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