Rivka Goldshleger

TISSUE-SPECIFIC DISTRIBUTION AND MODULATORY ROLE OF THE GAMMA SUBUNIT OF THE NA,K-ATPASE

The Na,K-ATPase comprises a catalytic α subunit and a glycosylated β subunit. Another membrane polypeptide, γ, first described by Forbush et al.(Forbush, B., III, Kaplan, J. H., and Hoffman, J. F. (1978)Biochemistry 17, 3667–3676) associates with α and β in purified kidney enzyme preparations. In this study, we have used a polyclonal anti-γ antiserum to define the tissue specificity and topology of γ and to address the question of whether γ has a functional role. The trypsin sensitivity of the amino terminus of the γ 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 γ 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, α1-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 γ function, the effects of the anti-γ serum on the kinetic behavior of rat kidney sodium pumps was examined. The following evidence suggests that γ stabilizes E1conformation(s) of the enzyme and that anti-γ counteracts this effect: (i) anti-γ inhibits Na,K-ATPase, and the inhibition increases at acidic pH under which condition the E2(K) → E1 phase of the reaction sequence becomes more rate-limiting, (ii) the oligomycin-stimulated increase in the level of phosphoenzyme was greater in the presence of anti-γ indicating that the antibody shifts the E1 ↔ ↔ E2P equilibria toward E2P, and (iii) when the Na+-ATPase reaction is assayed with the Na+concentration reduced to levels (≤2 mm) which limit the rate of the E1 → → E2P transition, anti-γ is stimulatory. These observations taken together with evidence that the pig γ 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 γ subunit is a tissue-specific regulator which shifts the steady-state equilibria toward E1. Accordingly, binding of anti-γ disrupts αβ-γ interactions and counteracts these modulatory effects of the γ subunit.

Evidence for an interaction between adducin and Na+-K+-ATPase: relation to genetic hypertension

Adducin point mutations are associated with genetic hypertension in Milan hypertensive strain (MHS) rats and in humans. In transfected cells, adducin affects actin cytoskeleton organization and increases the Na+-K+-pump rate. The present study has investigated whether rat and human adducin polymorphisms differently modulate rat renal Na+-K+-ATPase in vitro. We report the following. 1) Both rat and human adducins stimulate Na+-K+-ATPase activity, with apparent affinity in tens of nanomolar concentrations. 2) MHS and Milan normotensive strain (MNS) adducins raise the apparent ATP affinity for Na+-K+-ATPase. 3) The mechanism of action of adducin appears to involve a selective acceleration of the rate of the conformational change E2 (K) → E1 (Na) or E2(K) ⋅ ATP → E1Na ⋅ ATP. 4) Apparent affinities for mutant rat and human adducins are significantly higher than those for wild types. 5) Recombinant human α- and β-adducins stimulate Na+-K+-ATPase activity, as do the COOH-terminal tails, and the mutant proteins display higher affinities than the wild types. 6) The cytoskeletal protein ankyrin, which is known to bind to Na+-K+-ATPase, also stimulates enzyme activity, whereas BSA is without effect; the effects of adducin and ankyrin when acting together are not additive. 7) Pig kidney medulla microsomes appear to contain endogenous adducin; in contrast with purified pig kidney Na+-K+-ATPase, which does not contain adducin, added adducin stimulates the Na+-K+-ATPase activity of microsomes only about one-half as much as that of purified Na+-K+-ATPase. Our findings strongly imply the existence of a direct and specific interaction between adducin and Na+-K+-ATPase in vitro and also suggest the possibility of such an interaction in intact renal membranes.Adducin point mutations are associated with genetic hypertension in Milan hypertensive strain (MHS) rats and in humans. In transfected cells, adducin affects actin cytoskeleton organization and increases the Na(+)-K(+)-pump rate. The present study has investigated whether rat and human adducin polymorphisms differently modulate rat renal Na(+)-K(+)-ATPase in vitro. We report the following. 1) Both rat and human adducins stimulate Na(+)-K(+)-ATPase activity, with apparent affinity in tens of nanomolar concentrations. 2) MHS and Milan normotensive strain (MNS) adducins raise the apparent ATP affinity for Na(+)-K(+)-ATPase. 3) The mechanism of action of adducin appears to involve a selective acceleration of the rate of the conformational change E(2) (K) --> E(1) (Na) or E(2)(K). ATP --> E(1)Na. ATP. 4) Apparent affinities for mutant rat and human adducins are significantly higher than those for wild types. 5) Recombinant human alpha- and beta-adducins stimulate Na(+)-K(+)-ATPase activity, as do the COOH-terminal tails, and the mutant proteins display higher affinities than the wild types. 6) The cytoskeletal protein ankyrin, which is known to bind to Na(+)-K(+)-ATPase, also stimulates enzyme activity, whereas BSA is without effect; the effects of adducin and ankyrin when acting together are not additive. 7) Pig kidney medulla microsomes appear to contain endogenous adducin; in contrast with purified pig kidney Na(+)-K(+)-ATPase, which does not contain adducin, added adducin stimulates the Na(+)-K(+)-ATPase activity of microsomes only about one-half as much as that of purified Na(+)-K(+)-ATPase. Our findings strongly imply the existence of a direct and specific interaction between adducin and Na(+)-K(+)-ATPase in vitro and also suggest the possibility of such an interaction in intact renal membranes.

Functional Role and Immunocytochemical Localization of the γa and γb Forms of the Na,K-ATPase γ Subunit

The γ 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 γ is present primarily in the kidney as two main splice variants, γa and γb, 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 γa and γb. At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat α1-transfected HeLa cells, i.e. (i) an increase in apparent affinity for ATP, probably secondary to a left shift in E 1 ↔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-γ) abrogate the first effect but not the second. In contrast, γa and γb show differences in their localization along the kidney tubule. Using anti-γ (C-terminal) and antibodies to the rat α subunit as well as antibodies to identify cell types, double immunofluorescence showed γ in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both γa and γb. In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express γ but at lower levels. Antibodies specific for γa and γb showed differences in their cortical location; γa is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, γb but not γa is present in the cortical TAL only. Thus, the importance of γa and γb may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve.

Selectivity of Digitalis Glycosides for Isoforms of Human Na,K-ATPase

There are four isoforms of the α subunit (α1–4) and three isoforms of the β subunit (β1–3) of Na,K-ATPase, with distinct tissue-specific distribution and physiological functions. α2 is thought to play a key role in cardiac and smooth muscle contraction and be an important target of cardiac glycosides. An α2-selective cardiac glycoside could provide important insights into physiological and pharmacological properties of α2. The isoform selectivity of a large number of cardiac glycosides has been assessed utilizing α1β1, α2β1, and α3β1 isoforms of human Na,K-ATPase expressed in Pichia pastoris and the purified detergent-soluble isoform proteins. Binding affinities of the digitalis glycosides, digoxin, β-methyl digoxin, and digitoxin show moderate but highly significant selectivity (up to 4-fold) for α2/α3 over α1 (KD α1 > α2 = α3). By contrast, ouabain shows moderate selectivity (≈2.5-fold) for α1 over α2 (KD α1 ≤ α3 < α2). Binding affinities for the three isoforms of digoxigenin, digitoxigenin, and all other aglycones tested are indistinguishable (KD α1 = α3 = α2), 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 α2-selective. This supports a major role of α2 in cardiac contraction and cardiotonic effects of digitalis glycosides.

Purification of Na+,K+-ATPase Expressed in Pichia pastoris Reveals an Essential Role of Phospholipid-Protein Interactions

Na+,K+-ATPase (porcine α/his10-β) has been expressed in Pichia Pastoris, solubilized in n-dodecyl-β-maltoside and purified to 70–80% purity by nickel-nitrilotriacetic acid chromatography combined with size exclusion chromatography. The recombinant protein is inactive if the purification is done without added phospholipids. The neutral phospholipid, dioleoylphosphatidylcholine, preserves Na+,K+-ATPase activity of protein prepared in a Na+-containing medium, but activity is lost in a K+-containing medium. By contrast, the acid phospholipid, dioleoylphosphatidylserine, preserves activity in either Na+- or K+-containing media. In optimal conditions activity is preserved for about 2 weeks at 0 °C. Both recombinant Na+,K+-ATPase and native pig kidney Na+,K+-ATPase, dissolved in n-dodecyl-β-maltoside, appear to be mainly stable monomers (α/β) as judged by size exclusion chromatography and sedimentation velocity. Na+,K+-ATPase activities at 37 °C of the size exclusion chromatography-purified recombinant and renal Na+,K+-ATPase are comparable but are lower than that of membrane-bound renal Na+,K+-ATPase. The β subunit is expressed in Pichia Pastoris as two lightly glycosylated polypeptides and is quantitatively deglycosylated by endoglycosidase-H at 0 °C, to a single polypeptide. Deglycosylation inactivates Na+,K+-ATPase prepared with dioleoylphosphatidylcholine, whereas dioleoylphosphatidylserine protects after deglycosylation, and Na+,K+-ATPase activity is preserved. This work demonstrates an essential role of phospholipid interactions with Na+,K+-ATPase, including a direct interaction of dioleoylphosphatidylserine, and possibly another interaction of either the neutral or acid phospholipid. Additional lipid effects are likely. A role for the β subunit in stabilizing conformations of Na+,K+-ATPase (or H+,K+-ATPase) with occluded K+ ions can also be inferred. Purified recombinant Na+,K+-ATPase could become an important experimental tool for various purposes, including, hopefully, structural work.

Electrogenic and Electroneutral Transport Modes of Renal Na/K ATPase Reconstituted into Proteoliposomes

SummaryThis paper describes measurements of electrical potentials generated by renal Na/K-ATPase reconstituted into proteoliposomes, utilizing the anionic dye, oxonol VI. Calibration of absorption changes with imposed diffusion potentials allows estimation of absolute values of electrogenic potentials.ATP-dependent Nacyt/Kexc exchange in K-loaded vesicles generates large potentials, up to 250 mV. By comparing initial rates or steady-state potentials with ATP-dependent22Na fluxes in different conditions, it is possible to infer whether coupling ratios are constant or variable. For concentrations of Nacyt (2–50mm) and ATP (1–1000 μm) and pHs (6.5–8.5), the classical 3Nacyt/2Kexc coupling ratio is maintained. However, at low Nacyt concentrations (<0.8mm), the coupling ratio is apparently less than 3Nacyt/2Kexc.ATP-dependent Nacyt/congenerexc exchange in vesicles loaded with Rb, Cs, Li and Na is electrogenic. In this mode congeners, including Naexc, act as Kexc surrogates in an electrogenic 3Nacyt/2congenerexc exchange. (ATP+Pi)-dependent Kcyt/Kexc exchange in K-loaded vesicles is electroneutral.ATP-dependent “uncoupled” Na flux into Na- and K-free vesicles is electroneutral at pH 6.5–7.0 but becomes progressively electrogenic as the pH is raised to 8.5. The22Na flux shows no anion specificity. We propose that “uncoupled” Na flux is an electroneutral 3Nacyt/3Hexc exchange at pH 6.5–7.0 but at higher pHs the coupling ratio changes progressively, reaching 3Na/no ions at pH 8.5. Slow passive pump-mediated net K uptake into Na- and K-free vesicles is electroneutral, and may also involve Kcyt/Hexc exchange.We propose the general hypothesis that coupling ratios are fixed when cation transport sites are saturated, but at low concentrations of transported cations, e.g., Nacyt in Na/K exchange and Hexc in “uncoupled” Na flux, coupling ratios may change.

An Effect of Voltage on Binding of Na+ at the Cytoplasmic Surface of the Na+-K+ Pump

This work utilizes proteoliposomes reconstituted with renal Na+-K+-ATPase to study effects of electrical potential (40-80 mV) on activation of pump-mediated fluxes of Na+ or Rb+ (K+) ions and on inhibitory effects of Rb+ ions or organic cations. The latter include guanidinium derivatives that are competitive Na+-like antagonists (David, P., Mayan, H., Cohen. H., Tal, D. M., and Karlish, S. J. D.(1992) J. Biol. Chem. 267, 1141-1149). Cytoplasmic side-positive diffusion potentials significantly decreased the K(0.5) of Na+ at the cytoplasmic surface for activation of ATP-dependent Na+-K+ exchange but did not affect the inhibitory potency of Rb+ (K+) or any Na+-like antagonist. Diffusion potentials did not affect activation of Rb+-Rb+ exchange by Rb+ ions at the cytoplasmic surface and had only a minor effect on Rb+ activation at the extracellular surface. Previously, we proposed that the cation binding domain consists of two negatively charged sites, to which two K+ or two Na+ ions bind, and one neutral site for the third Na+ (Glynn, I. M., and Karlish, S. J. D.(1990) Annu. Rev. Biochem. 59, 171-205). The present experiments suggest that binding of a Na+ ion in the neutral site at the cytoplasmic surface is sensitive to voltage. By contrast, binding of Rb+ ions at the extracellular surface of renal pumps appears to be only weakly or insignificantly affected by voltage. Inferences on the identity of the charge-carrying steps, based on experiments using proteoliposomes, are discussed in relation to recent evidence that dissociation of Na+ or association of K+ ions, at the extracellular surface, represent the major charge-carrying steps.

The Energy Transduction Mechanism of Na,K-ATPase Studied with Iron-catalyzed Oxidative Cleavage

This paper extends our recent report on specific iron-catalyzed oxidative cleavages of renal Na,K-ATPase and effects ofE 1 ↔ E 2conformational transitions (Goldshleger, R., and Karlish, S. J. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9596–9601). The experiments indicate that only peptide bonds close to a bound Fe2+ ion are cleaved, and provide evidence on proximity of the different cleavage positions in the native enzyme. A sequence HFIH near trans-membrane segment M3 appears to be involved in Fe2+ binding. Previously we hypothesized that E 2 and E 1conformations are characterized by formation or relaxation of interactions within the α subunit at or near highly conserved sequences, TGES in the minor cytoplasmic loop and CSDK, MVTGD, and VNDSPALKK in the major cytoplasmic loop. This concept has been tested by examining iron-catalyzed cleavage in both non-phosphorylated and phosphorylated conformations and effects of phosphate, vanadate, and ouabain. The results imply that both E 1 ↔E 2 and E 1P ↔E 2P transitions are indeed associated with formation and relaxation of interactions between cytoplasmic domains, comprising the minor loop plus N-terminal tail leading into M1 and major loop, respectively. Furthermore, it appears that either non-covalently or covalently bound phosphate bind near CSDK and MVTGD, and Mg2+ ions may bind to residues within TGES and VNDSPALKK and to bound phosphate. Thus cytoplasmic domain interactions seem to occur within or near the active site. We discuss the relationship between structural changes in the cytoplasmic domain and movements of trans-membrane segments that lead to cation transport. Presumably conformation-dependent formation and relaxation of domain interactions underlie energy transduction in all P-type pumps.

Structural Organization and Energy Transduction Mechanism of Na+,K+-ATPase Studied with Transition Metal-Catalyzed Oxidative Cleavage

This chapter describes contributions of transition metal-catalyzed oxidative cleavage of Na+,K+-ATPase to our understanding of structure–function relations. In the presence of ascorbate/H2O2, specific cleavages are catalyzed by the bound metal and because more than one peptide bond close to the metal can be cleaved, this technique reveals proximity of the different cleavage positions within the native structure. Specific cleavages are catalyzed by Fe2+ bound at the cytoplasmic surface or by complexes of ATP–Fe2+, which directs the Fe2+ to the normal ATP–Mg2+ site. Fe2+- and ATP–Fe2+-catalyzed cleavages reveal large conformation-dependent changes in interactions between cytoplasmic domains, involving conserved cytoplasmic sequences, and a change of ligation of Mg2+ ions between E1P and E2P, which may be crucial in facilitating hydrolysis of E2P. The pattern of domain interactions in E1 and E2 conformations, and role of Mg2+ ions, may be common to all P-type pumps. Specific cleavages can also be catalyzed by Cu2+ ions, bound at the extracellular surfaces, or a hydrophobic Cu2+-diphenyl phenanthroline (DPP) complex, which directs the Cu2+ to the membrane–water interface. Cu2+- or Cu2+-DPP-catalyzed cleavages are providing information on α/β subunit interactions and spatial organization of transmembrane segments. Transition metal-catalyzed cleavage could be widely used to investigate other P-type pumps and membrane proteins and, especially, ATP binding proteins.

Characterization of Disulfide Cross-links between Fragments of Proteolyzed Na,K-ATPase IMPLICATIONS FOR SPATIAL ORGANIZATION OF TRANS-MEMBRANE HELICES

This study characterizes disulfide cross-links between fragments of a well defined tryptic preparation of Na,K-ATPase, 19-kDa membranes solubilized with C12E10in conditions preserving an intact complex of fragments and Rb occlusion (Or, E., Goldshleger, R., Tal, D. M., and Karlish, S. J. D. (1996) Biochemistry 35, 6853–6864). Upon solubilization, cross-links form spontaneously between the β subunit, 19- and 11.7-kDa fragments of the α subunit, containing trans-membrane segments M7-M10 and M1/M2, respectively. Treatment with Cu2+-phenanthroline (CuP) improves efficiency of cross-linking. Sequencing and immunoblot analysis have shown that the cross-linked products consist of a mixture of β-19 kDa dimers (≈65%) and β-19 kDa–11.7 kDa trimers (≈35%). The α-β cross-link has been located within the 19-kDa fragment to a 6.5-kDa chymotryptic fragment containing M8, indicating that βCys44 is cross-linked to either Cys911 or Cys930. In addition, an internal cross-link between M9 and M10, Cys964-Cys983, has been found by sequencing tryptic fragments of the cross-linked product. The M1/M2-M7/M10 cross-link has not been identified directly. However, we propose that Cys983 in M10 is cross-linked either to Cys104 in M1 or internally to Cys964 in M9. Based on this study, cross-linking induced byo-phthalaldehyde (Or, E., Goldshleger, R., and Karlish, S. J. D. (1998) Biochemistry 37, 8197–8207), and information from the literature, we propose an approximate spatial organization of trans-membrane segments of the α and β subunits.