Steven J. D. Karlish

Characterization of conformational changes in (Na,K) ATPase labeled with fluorescein at the active site

Conformational changes have been studied in (Na,K) ATPase labeled at or near the ATP binding region with fluorescein following incubation with fluorescein isothiocyanate (FITC). One or two fluorescein groups are bound per ATPase molecule. (Na,K) ATPase activity, phosphorylation from ATP, and nucleotide binding are abolished in labeled enzyme, but phosphorylation from inorganic phosphate or K-phosphatase activity are only partially inactivated. The fluorescein groups are incorporated only into the 96 KD catalytic chain of the (Na,K) ATPase, and presence of ATP during the incubation with FITC protects against the incorporation and inhibition of enzymic activity. Upon trypsin treatment of labeled membranes the fluorescein appears first in a 58 KD fragment and eventually is released into the medium. The fluorescein-labeled (Na,K) ATPase shows a large quenching of fluorescence (15–20%) on conversion of the E1 or E1 · Na conformation in cation-free or Na+-rich media to the E2 · (K) form in K+ (or congeners Tl+, Rb+, Cs+, NH4+) rich media. Cation titrations suggest that K+ and Na+ ions compete at a single binding site and stabilize E1 · Na or E2 · (K) respectively;KK≈0.23 mM,KNa≈1.2 mM. The rate of the conformational transition E2 · (K) → E1 · Na is slow,k=0.3 sec−1, but contrary to previous experience [7, 8] ATP does not stimulate this rate. The rate of the transitions E1 + K+ → E2 · (K) rises sharply with K+ concentration and shows saturation behavior, from which akmax≈286 sec−1 andKk≈74 mM are deduced. The data support and extend the previous suggestion that K+ ions bound initially at a low-affinity (probably cytoplasm oriented) site in state E1 are trapped in the occluded form E2 · (K) by the conformational change poised far (Kc≈1000) in the direction of E2 · (K). It is proposed in addition that at least two binding sites for K+ exist at the cytoplasmic surface of isolated (Na,K) ATPase in state E1 but a large difference in affinities precludes detection in fluorescence titrations of more than one site. A variety of ligands in addition to K+ produce fluorescence-quenched or E2 forms of the labeled (Na,K) ATPase. These include Mg2+ plus inorganic phosphate, without or with K+ ions (E2P or E2P · K) or with ouabain (E2-ouabain or E2P · ouabain). Na+ ions antagonize these effects. The collected data support the notion that there may be many subspecies of the E1 and E2 forms (either phosphorylated or nonphosphorylated) with different numbers of Na+ and/or K+ ions bound or occluded, each subspecies having a characteristic ability to catalyze reactions and/or transport cations. The relationship between the conformational changes in fluorescein-labeled enzyme and the subunit structure of the (Na,K) ATPase is discussed with particular reference to “half of the site” models for ATP hydrolysis.

Phospholemman (FXYD1) associates with Na,K-ATPase and regulates its transport properties

A family of small, single-span membrane proteins (the FXYD family) has recently been defined based on their sequence and structural homology. Some members of this family have already been identified as tissue-specific regulators of Na,K-ATPase (NKA). In the present study, we demonstrate that phospholemman (PLM) (FXYD1), so far considered to be a heart- and muscle-specific channel or channel-regulating protein, associates specifically and stably with six different α-β isozymes of NKA after coexpression in Xenopus oocytes, and with α1–β, and less efficiently with α2–β isozymes, in native cardiac and skeletal muscles. Stoichiometric association of PLM with NKA occurs posttranslationally either in the Golgi or the plasma membrane. Interaction of PLM with NKA induces a small decrease in the external K+ affinity of α1–β1 and α2–β1 isozymes and a nearly 2-fold decrease in the internal Na+ affinity. In conclusion, this study demonstrates that PLM is a tissue-specific regulator of NKA that may play an essential role in muscle contractility.

Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism.

1. The protein fluorescence intensity of (Na+ + K+)-ATPase is enhanced following binding of K+ at low concentrations. The properties of the response suggest that one or a few tryptophan residues are affected by a conformational transition between the K-bound form E2 . (K) and a Na-bound form E1 . Na. 2. The rate of the conformational transition E2 . (K) leads to E . Na has been measured with a stopped-flow fluorimeter by exploiting the difference in fluorescence of the two states. In the absence of ATP the rate is very slow, but it is greatly accelerated by binding of ATP to a low affinity site. 3. Transient changes in tryptophan fluorescence accompany hydrolysis of ATP at low concentrations, in media containing Mg2+, Na+ and K+. The fluorescence response reflects interconversion between the initial enzyme conformation, E1 . Na and the steady-state turnover intermediate E2 . (K). 4. The phosphorylated intermediate, E2P can be detected by a fluorescence increase accompanying hydrolysis of ATP in media containing Mg2+ and Na+ but no K+. 5. The conformational states and reaction mechanism of the (Na+ + K+)-ATPase are discussed in the light of this work. The results permit a comparison of the behaviour of the enzyme at both low and high nucleotide concentrations.

Conformational transitions between Na+-bound and K+-bound forms of (Na+ + K+)-ATPase, studied with formycin nucleotides

1. Fluorescence measurements have shown that formycin triphosphate (FTP) or formycin diphosphate (FDP) bound to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in Na+-containing media can be displaced by the following ions (listed in order of effectiveness): Tl+, K+, Rb+, NH4+, Cs+. 2. The differences between the nucleotide affinities displayed by the enzyme in predominantly Na+ and predominantly K+ media in the absence of phosphorylation, are thought to reflect changes in enzyme conformation. These changes can therefore be monitored by observing the changes in fluorescence that accompany net binding or net release of formycin nucleotides. 3. The transition from a K+-bound form (E2-(K)) to an Na+-bound form (E1-Na) is remarkably slow at low nucleotide concentrations, but is accelerated if the nucleotide concentration is increased. This suggests that the binding of nucleotide to a low-affinity site on E2-(K) accelerates its conversion to E1-Na; it supports the hypothesis that during the normal working of the pump, ATP, acting at a low affinity site, accelerates the conversion of dephosphoenzyme, newly formed by K+-catalysed hydrolysis of E2P, to a form in which it can be phosphorylated in the presence of Na+. 4. The rate of the reverse transformation, E1-Na to E2-(K), varies roughly linearly with the K+ concentration up to the highest concentration at which the rate can be measured (15 mM). Since much lower concentrations of K+ are sufficient to displace the equilibrium to the K-form, we suggest that the sequence of events is: (i) combination of K+ with low affinity (probably internal) binding sites, followed by (ii) spontaneous conversion of the enzyme to a form, E2-(K), containing occluded K+. 5. Mg2+ or oligomycin slows the rate of conversion of E1-Na to E2-(K) but does not significantly affect the rate of conversion of E2-(K) to E1-Na. 6. In the light of these and previous findings, we propose a model for the sodium pump in which conformational changes alternate with trans-phosphorylations, and the inward and outward fluxes of both Na+ and K+ each involve the transfer of a phosphoryl group as well as a change in conformation between E1 and E2 forms of the enzyme or phosphoenzyme.

Indications for an oligomeric structure and for conformational changes in sarcoplasmic reticulum Ca2+-ATPase labelled selectively with fluorescein

Fluorescein isothiocyanate is a highly specific inhibitor of the Ca2+-ATPase from sarcoplasmic reticulum. The Ca2+ pumping is inhibited completely at a fluorescein isothiocyanate concentration half that of the ATPase protein, indicating that the protein is at least a dimer. ATP protected specifically against fluorescein isothiocyanate inhibition, indicating that fluorescein isothiocyanate may react at the nucleotide binding site of the ATPase (probably with a reactive lysine residue). The fluorescein is incorporated almost exclusively into the 105 kdalton catalytic polypeptide of the ATPase and digestion by trypsin gives rise to a fluorescein-labelled 45 kdalton fragment. Conformational changes induced by addition of Ca can be studied conveniently with the fluorescein-labelled ATPase.

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.

Expression and functional role of the gamma subunit of the Na, K-ATPase in mammalian cells.

The functional role of the γ subunit of the Na,K-ATPase was studied using rat γ cDNA-transfected HEK-293 cells and an antiserum (γC33) specific for γ. Although the sequence for γ was verified and shown to be larger (7237 Da) than first reported, it still comprises a single initiator methionine despite the expression of a γC33-reactive doublet on immunoblots. Kinetic analysis of the enzyme of transfected compared with control cells and of γC33-treated kidney pumps shows that γ regulates the apparent affinity for ATP. Thus, γ-transfected cells have a decreasedK′ATP as shown in measurements of (i)K′ATP of Na,K-ATPase activity and (ii) K+ inhibition of Na-ATPase at 1 μm ATP. Consistent with the behavior of γ-transfected cells, γC33 pretreatment increases K′ATP of the kidney enzyme and K+ inhibition (1 μm ATP) of both kidney and γ-transfected cells. These results are consistent with previous findings that an antiserum raised against the pig γ subunit stabilizes the E 2(K) form of the enzyme (Therien, A. G., Goldshleger, R., Karlish, S. J., and Blostein, R. (1997) J. Biol. Chem. 272, 32628–32634). Overall, our data demonstrate that γ 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-γ antiserum.

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.