Metal fluoride complexes of Na,K-ATPase: characterization of fluoride-stabilized phosphoenzyme analogues and their interaction with cardiotonic steroids

The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K(+)·P(i) with ouabain bound with apparently low affinity (1). In the present work inhibition, and subsequent reactivation by high Na(+), after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na(+) demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway.     

A chimeric gastric H+,K+-ATPase inhibitable with both ouabain and SCH 28080

2-Methyl-8-(phenylmethoxy)imidazo(1,2-a)pyridine-3acetonitrile+ ++ (SCH 28080) is a K+ site inhibitor specific for gastric H+,K+-ATPase and seems to be a counterpart of ouabain for Na+,K+-ATPase from the viewpoint of reaction pattern (i.e. reversible binding, K+ antagonism, and binding on the extracellular side). In this study, we constructed several chimeric molecules between H+,K+-ATPase and Na+,K+-ATPase alpha-subunits by using rabbit H+,K+-ATPase as a parental molecule. We found that the entire extracellular loop 1 segment between the first and second transmembrane segments (M1 and M2) and the luminal half of the M1 transmembrane segment of H+, K+-ATPase alpha-subunit were exchangeable with those of Na+, K+-ATPase, respectively, preserving H+,K+-ATPase activity, and that these segments are not essential for SCH 28080 binding. We found that several amino acid residues, including Glu-822, Thr-825, and Pro-829 in the M6 segment of H+,K+-ATPase alpha-subunit are involved in determining the affinity for this inhibitor. Furthermore, we found that a chimeric H+,K+-ATPase acquired ouabain sensitivity and maintained SCH 28080 sensitivity when the loop 1 segment and Cys-815 in the loop 3 segment of the H+,K+-ATPase alpha-subunit were simultaneously replaced by the corresponding segment and amino acid residue (Thr) of Na+,K+-ATPase, respectively, indicating that the binding sites of ouabain and SCH 28080 are separate. In this H+, K+-ATPase chimera, 12 amino acid residues in M1, M4, and loop 1-4 that have been suggested to be involved in ouabain binding of Na+, K+-ATPase alpha-subunit are present; however, the low ouabain sensitivity indicates the possibility that the sensitivity may be increased by additional amino acid substitutions, which shift the overall structural integrity of this chimeric H+,K+-ATPase toward that of Na+,K+-ATPase.     

Nerve Growth Factor Induces Na+, K+-ATPase in a Nerve Cell Line

The effects of nerve growth factor (NGF) on induction of Na+,K+-ATPase were examined in a rat pheochromocytoma cell line, PC12h. Na+,K+-ATPase activity in a crude particulate fraction from the cells increased from 0.37 +/- 0.02 (n = 19) to 0.55 +/- 0.02 (n = 20) (means +/- SEM, mumol Pi/min/mg of protein) when cultured with NGF for 5-11 days. The increase caused by NGF was prevented by addition of specific anti-NGF antibodies. Epidermal growth factor and insulin had only a small effect on induction of Na+,K+-ATPase. A concentration of basic fibroblast growth factor three times higher than that of NGF showed a similar potency to NGF. The molecular form of the enzyme was judged as only the alpha form in both the untreated and the NGF-treated cells by a simple pattern of low-affinity interaction with cardiotonic steroids: inhibition of enzyme activity by strophanthidin (Ki approximately 1 mM) and inhibition of Rb+ uptake by ouabain (Ki approximately 100 microM). As a consequence, during differentiation of PC12h cells to neuron-like cells, NGF increases the alpha form of Na+,K+-ATPase, but does not induce the alpha(+) form of the enzyme, which has a high sensitivity for cardiotonic steroid and is a characteristic form in neurons.     

Two classes of ouabain receptors in chick ventricular cardiac cells and their relation to (Na+,K+)-ATPase inhibition, intracellular Na+ accumulation, Ca2+ influx, and cardiotonic effect

The biochemical and pharmacological properties of the (Na+,K+)-ATPase have been studied at different stages of chick embryonic heart development in ovo and under cell culture conditions. The results show the existence of two families of ouabain binding sites: a low affinity binding site with a dissociation constant (Kd) of 2-6 microM for the ouabain-receptor complex and a high affinity binding site with a Kd of 26-48 nM. Levels of high affinity sites gradually decrease during cardiac ontogenesis to reach a plateau near 14 days of development. Conversely the number of low affinity binding sites is essentially invariant between 5 days and hatching. Cultured cardiac cells display the same binding characteristics as those found in intact ventricles. Inhibition of 86Rb+ uptake in cultured cardiac cells and an increase in intracellular Na+ concentration, due to (Na+,K+)-ATPase blockade, occur in a ouabain concentration range corresponding to the saturation of the low affinity ouabain site. Ouabain-stimulated 45Ca2+ uptake increases in parallel with the increase in the intracellular Na+ concentration. It is suppressed in Na+-free medium or when Na+ is replaced by Li+ suggesting that the increase is due to the indirect activation of the Na+/Ca2+ exchange system in the plasma membrane. Dose-response curves for the inotropic effects of ouabain on papillary muscle and on ventricular cells in culture indicate that the development of the cardiotonic properties is parallel to the saturation of the low affinity binding site for ouabain. Therefore, inhibition of the cardiac (Na+,K+)-ATPase corresponding to low affinity ouabain binding sites seems to be responsible for both the cardiotonic and cardiotoxic effects of the drug.     

Phe783, Thr797, and Asp804 in transmembrane hairpin M5-M6 of Na+,K+-ATPase play a key role in ouabain binding

Ouabain is a glycoside that binds to and inhibits the action of Na+,K+-ATPase. Little is known, however, about the specific requirements of the protein surface for glycoside binding. Using chimeras of gastric H+,K+-ATPase and Na+,K+-ATPase, we demonstrated previously that the combined presence of transmembrane hairpins M3-M4 and M5-M6 of Na+,K+-ATPase in a backbone of H+,K+-ATPase (HN34/56) is both required and sufficient for high affinity ouabain binding. Since replacement of transmembrane hairpin M3-M4 by the N terminus up to transmembrane segment 3 (HNN3/56) resulted in a low affinity ouabain binding, hairpin M5-M6 seems to be essential for ouabain binding. To assess which residues of M5-M6 are required for ouabain action, we divided this transmembrane hairpin in seven parts and individually replaced these parts by the corresponding sequences of H+,K+-ATPase in chimera HN34/56. Three of these chimeras failed to bind ouabain following expression in Xenopus laevis oocytes. Altogether, these three chimeras contained 7 amino acids that were specific for Na+,K+-ATPase. Individual replacement of these 7 amino acids by the corresponding amino acids in H+,K+-ATPase revealed a dramatic loss of ouabain binding for F783Y, T797C, and D804E. As a proof of principle, the Na+,K+-ATPase equivalents of these 3 amino acids were introduced in different combinations in chimera HN34. The presence of all 3 amino acids appeared to be required for ouabain action. Docking of ouabain onto a three-dimensional-model of Na+,K+-ATPase suggests that Asp804, in contrast to Phe783 and Thr797, does not actually form part of the ouabain-binding pocket. Most likely, the presence of this amino acid is required for adopting of the proper conformation for ouabain binding.     

Ouabain is a potent promoter of growth and activator of ERK1/2 in ouabain-resistant rat renal epithelial cells

Endogenous cardiotonic steroids (ECS) are putative ligands of the inhibitory binding site of the membrane sodium pump (Na+, K+-ATPase). There is growing evidence that cardiotonic steroids may promote the growth of cardiac and vascular myocytes, including evidence indicating growth stimulation at concentrations in the same range as circulating ECS concentrations. We investigated four parameters to determine whether ouabain, a proposed ECS, promotes growth of immortalized rat proximal tubule epithelial cells: cell count by hemocytometer; metabolic activity as reflected in the mitochondrial conversion of the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, to its formazan product (MA); DNA synthesis reflected as bromodeoxyuridine incorporation (DNA); and mitosis reflected as histone phosphorylation state detected using anti-phosphohistone 3 antibody (HP). Maximum stimulatory responses were observed at 1 nm ouabain (MA, 20.3% increase, p < 0.01; DNA, 28.4% increase, p < 0.001; HP, maximum response at 0.5 h, 50% increase, p < 0.001). We observed that growth stimulation was associated with stimulation of ERK1/2 phosphorylation (ERK-P), and both growth and ERK-P could be blocked by the MEK inhibitor (U0126, 100 nm). Western blot analysis revealed that the only alpha isoform of Na+, K+-ATPase that could be detected in these cultures was the highly ouabain-resistant alpha1 isoform. Measurement of ouabain inhibition of ion transport in these cultures using 86Rb+ uptake revealed the predominance of the expected ouabain-resistant isoform (IC50 = 24 microm) and an additional minor ( approximately 15%) ouabain-sensitive inhibition with IC50 approximately 30 pm. Similar bimodal transport inhibition curves were obtained in freshly dissected rat proximal tubules. These results indicate that renal epithelial cells may be a sensitive target of the ERK1/2-activating and growth-promoting effects of ouabain even in the presence of ouabain-resistant Na+, K+-ATPase.     

Visualizing the mapped ion pathway through the Na,K-ATPase pump

The Na+,K+-ATPase pump achieves thermodynamically uphill exchange of cytoplasmic Na+ ions for extracellular K+ ions by using ATP-mediated phosphorylation, followed by autodephosphorylation, to power conformational changes that allow ion access to the pump's binding sites from only one side of the membrane at a time. Formally, the pump behaves like an ion channel with two tightly coupled gates that are constrained to open and close alternately. The marine agent palytoxin disrupts this coupling, allowing both gates to sometimes be open, so temporarily transforming a pump into an ion channel. We made a cysteine scan of Na+,K+-ATPase transmembrane (TM) segments TM1 to TM6, and used recordings of Na+ current flow through palytoxin-bound pump-channels to monitor accessibility of introduced cysteine residues via their reaction with hydrophilic methanethiosulfonate (MTS) reagents. To visualize the open-channel pathway, the reactive positions were mapped onto a homology model of Na+,K+-ATPase based on the structure of the related sarcoplasmic- and endoplasmic-reticulum (SERCA) Ca2+-ATPase in a BeF3--trapped state1,2, in which the extra-cytoplasmic gate is wide open (although the cytoplasmic access pathway is firmly shut). The results revealed a single unbroken chain of reactive positions that traverses the pump from the extracellular surface to the cytoplasm, comprises residues from TM1, TM2, TM4, and TM6, and passes through the equivalent of cation binding site II in SERCA, but not through site I. Cavity search analysis of the homology model validated its use for mapping the data by yielding a calculated extra-cytoplasmic pathway surrounded by MTS-reactive residues. As predicted by previous experimental results, that calculated extra-cytoplasmic pathway abruptly broadens above residue T806, at the outermost end of TM6 which forms the floor of the extracellular-facing vestibule. These findings provide a structural basis for further understanding cation translocation by the Na+,K+-ATPase and by other P-type pumps like the Ca2+- and H+,K+-ATPases.     

Cation sidedness in the phosphorylation step of Na+/K+-ATPase

Na+/K+ -ATPase, reconstituted into phospholipid vesicles, has been used to study the localisation of binding sites of ligands involved in the phosphorylation reaction. Inside-out oriented Na+/K+ -ATPase molecules are the only population in this system, which can be phosphorylated, as the rightside-out oriented as well as the non-incorporated enzyme molecules are inhibited by ouabain. In addition, the right-side-out oriented Na+/K+ -ATPase molecules have their ATP binding site intravesicularly and are thus not accessible to substrate added to the extravesicular medium. Functional binding sites for the following ligands have been demonstrated: (i) Potassium, acting at the extracellular side with high affinity (stimulating the dephosphorylation rate of the E2P conformation) and low affinity (inducing the non-phosphorylating E2K complex). (ii) Potassium, acting at the cytoplasmic side with both high and low affinity. The latter sites are also responsible for the formation of an E2K complex and complete with Na+ for its binding sites. (iii) Sodium at the cytoplasmic side responsible for stimulation of the phosphorylation reaction. (iv) Sodium (and amine buffers) at the extracellular side enhancing the phosphorylation level of Na+/K+ -ATPase where choline chloride has no effect. (v) Magnesium at the cytoplasmic side, stimulating the phosphorylation reaction and inhibiting it above optimal concentrations.     

Affinity chromatography for human Na+, K+-ATPase inhibitors in plasma and urine

Semi-purified dog kidney Na+,K+-ATPase cross-linked with ovalbumin was used in batch-wise affinity chromatography for the detection of endogenous Na+,K+-ATPase inhibitor in human plasma and urine. Ammonium acetate 1 M washed off the endogenous inhibitor from the immobilized enzyme. The inhibitory activity of the eluate from hypertensive plasma and urine was significantly higher (p less than 0.0025, n = 5 and p less than 0.005, n = 6 respectively) than that of normotensive. This latter was correlated with the ability of plasma from the same subjects to compete with ouabain binding to erythrocytes. Plasma and urine extracts inhibited the activity of Na+, K+-ATPase in a dose-dependent manner as ouabain does and were shown to contain 3 or 4 active compounds by high pressure liquid chromatography. The activity of some of these compounds was lost after peptidase treatment. These data support the heterogeneity of endogenous inhibitors of Na+,K+-ATPase activity in plasma and urine.     

Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+,K(+)-ATPase.

Site-specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the alpha 1-subunit of the ouabain resistant rat kidney Na+,K(+)-ATPase. cDNAs encoding either of the Na+,K(+)-ATPase mutants Pro328-->Ala and Leu332-->Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 microM ouabain were isolated, and the Na+,K+, ATP and pH dependencies of the Na+,K(+)-ATPase activity measured in the presence of 10 microM ouabain were analysed. Under these conditions the exogenous expressed Na+,K(+)-ATPase contributed more than 95% of the Na+,K(+)-ATPase activity. The Pro328-->Ala mutant displayed a reduced apparent affinity for Na+ (K0.5 (Na+) 13.04 mM), relative to the wild type (K0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu332-->Ala mutant was increased (K0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K0.5 (K+) 2.46 mM for Pro328-->Ala and 1.97 mM for Leu332-->Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K0.5 (ATP) 0.086 mM for Pro328-->Ala and 0.042 mM for Leu332-->Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)-->E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed.     

Specific sodium-22 binding to a purified sodium + potassium adenosine triphosphatase. Inhibition by ouabain.

Analysis of sodium-22 binding to purified sodium + potassium ion-activated adenosine triphosphatase (Na+, K+)-ATPase reveals the presence of two classes of binding sites. The higher affinity site (Kd = 0.2 mM) binds 6 to 7 nmol of sodium per mg of protein. Pretreatment of (Na+, K+)-ATPase with ouabain blocks the binding of sodium to this higher affinity site. Neither heat-denatured enzyme nor phospholipids extracted from the (Na+, K+)-ATPase contain a ouabain-inhibitable, higher affinity sodium binding site. The ouabain enzyme complex therefore appears to contain altered binding sites for cations.     

The effect of the distribution of polar groups in a steroid molecule on its inhibition of Na+,K+-ATPase

Transformed steroids having oxidized side chains in the D ring site and varying by polarity of the substituent at the ring A C(3)-position--acetates, glucosides or with free hydroxyls--in the concentration range 1 X 10(-4) - 1 X 10(-7) M were found to inhibit Na+, K+-ATPase. The extent of inhibition decreases with the rise in the polarity of the A region of the steroid molecule. With the compound devoid of polar groups in the D region an increase in the inhibitory activity is observed on passing from 3-acetate to 3-glucoside. The data obtained confirm the relationship between the extent of Na+, K+-ATPase inhibition and biphilicity of the molecule.     

The Na+ pump and intracellular signaling mechanisms

The main properties of Na+ /K(+)-ATPase as a natural receptor for cardiotonic steroids have been discusses. Primary attention is focused on structural and functional differences between the alpha-subunit isoforms of Na+/K(+)-ATPase in different tissues. General information on the role of the Na pump in signaling cascades in kidney epithelial cells, cardiomyocytes and neurons is presented. The data obtained indicate that, in neurons, several alpha-isoforms of Na+/K(+)-ATPase possessing different sensitivity to ouabain may have different signaling functions.     

Ligand binding sites of the ouabain-complexed (Na+ + K+)-ATPase

To clarify the mechanism of inhibition of (Na+ + K+)-ATPase by cardiac glycosides, we tried to see if ouabain binding alters the properties of the binding sites for Na+, K+, and ATP. Ouabain was bound in the presence of either Na+ + MgATP or MgPi. Ligand-induced changes in the rate of release of ouabain from the two resulting complexes were used as signals to determine the affinities, the numbers, and the interactions of the ligand binding sites. Because the two complexes showed differences in the properties of their ligand binding sites, and since neither complex could be converted to the other, it is concluded that either the enzyme has two dissimilar but mutually exclusive ouabain sites or that it can be frozen in two distinct conformations by ouabain. The following ligand sites were identified on the two complexes: 1) two coexisting ATP sites (K0.5 values, 0.1 and 2 mM) representing altered states of the catalytic and the regulatory sites of the native enzyme; 2) mutually exclusive Na+ and K+ sites whose affinities (K0.5 values, 1.3 mM Na+ and 0.1 mM K+) suggested their identities with the high affinity uptake sites of the native enzyme; and 3) coexisting low affinity Na+ and K+ sites (K0.5 values, 0.2-0.6 M) representing either the discharge sites, or the regulatory sites, or the access channels of the native enzyme. The data suggest that the inability of the ouabain-complexed enzyme to participate in the normal reaction cycle is not because of its lack of ligand binding sites but most likely due to ouabain-induced disruptions of interprotomer site-site interactions.     

[3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts

The interaction of the cardiac glycoside [3H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [3H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [3H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high- affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [3H]ouabain binding rates. Failure of 5'-adenylyl-beta- gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor.     

Insulin stimulation of (Na+,K+)-adenosine triphosphatase-dependent 86Rb+ uptake in rat adipocytes

Insulin stimulated the uptake of 86Rb+ (a K+ analog) in rat adipocytes and increased the steady state concentration of intracellular potassium. Half-maximal stimulation occurred at an insulin concentration of 200 pM. Both basal- and insulin-stimulated 86Rb+ transport rates depended on the concentration of external K+, external Na+, and were 90% inhibited by 10(-3) M ouabain and 10(-3) M KCN, indicating that the hormone was activating the (Na+,K+)-ATPase. Insulin had no effect on the entry of 22Na+ or exit of 86Rb+. Kinetic analysis demonstrated that insulin acted by increasing the maximum velocity, Vmax, of 86Rb+ entry. Inhibition of the rate of Rb+ uptake by ouabain was best described by a biphasic inhibition curve. Scatchard analysis of ouabain binding to intact cells indicated binding sites with multiple affinities. Only the rubidium transport sites which exhibited a high affinity for ouabain were stimulated by insulin. Stimulation required insulin binding to an intact cell surface receptor, as it was reversible by trypsinization. We conclude that the uptake of 86Rb+ by the (Na+,K+)-ATPase is an insulin-sensitive membrane transport process in the fat cell.     

Quantitative structure-activity relationships of cardiotonic steroids using empirical molecular electrostatic potentials and semiempirical molecular orbital calculations

Empirical molecular electrostatic potentials and a rigid receptor H atom model are used to calculate differences in the interaction energy of cardiotonic steroids with the digitalis receptor. An attempt is made to map the receptor H binding sites for two different steroid conformations with respect to 17 beta side-chain orientation. The calculated interaction energies using single-crystal X-ray structure data indicate linear relationships with the Na+, K+-ATPase inhibitory activity. On the basis of these correlations, the activity of 8 so far pharmacologically not investigated steroids containing C = O in 17 beta substituents are estimated with the help of structural data determined by means of the semiempirical CNDO molecular orbital theory.     

Effects of Ca2+ on the sodium pump observed in cardiac myocytes isolated from guinea pigs

It is presently unknown whether Ca2+ plays a role in the physiological control of Na+/K+-ATPase or sodium pump activity. Because the enzyme is exposed to markedly different intra- and extracellular Ca2+ concentrations, tissue homogenates or purified enzyme preparations may not provide pertinent information regarding this question. Therefore, the effects of Ca2+ on the sodium pump were examined with studies of [3H]ouabain binding and 86Rb+ uptake using viable myocytes isolated from guinea-pig heart and apparently maintaining ion gradients. In the presence of K+, a reduction of the extracellular Ca2+ increased specific [3H]ouabain binding observed at apparent binding equilibria: a half-maximal stimulation was observed when extracellular Ca2+ was lowered to about 50 microM. The change in [3H]ouabain binding was caused by a change in the number of binding sites accessible by ouabain instead of a change in their affinity for the glycoside. Ouabain-sensitive 86Rb+ uptake was increased by a reduction of extracellular Ca2+ concentration. Benzocaine in concentrations reported to reduce the rate of Na+ influx failed to influence the inhibitory effect of Ca2+ on glycoside binding. When [3H]ouabain binding was at equilibrium, the addition of Ca2+ decreased and that of EGTA increased the glycoside binding. Mn2+, which does not penetrate the cell membrane, had effects similar to Ca2+. In the absence of K+, cells lose their tolerance to Ca2+. Reducing Ca2+ concentration prevented the loss of rod-shaped cells but failed to affect specific [3H]ouabain binding observed in the absence of K+. These results indicate that a large change in extracellular Ca2+ directly affects the sodium pump in cardiac myocytes isolated from guinea pigs.     

Biological implication of conformational flexibility in ouabain: observations with two ouabain phosphate isomers

Ouabain is a highly polar and unusually potent sodium pump inhibitor that possesses uncommon conformational flexibility in its steroid A-ring moiety. The biological significance of ring flection in the cardiotonic steroids has not been described. Accordingly, we prepared ouabain 1,5,19- and 1,11,19-phosphates. The former stabilizes the steroid A-ring chair conformation and the latter locks the A-ring in the half-boat conformation and decreases flection of the ABC-ring moiety. Using a dog kidney cell line (MDCK) in a pH microphysiometer (Cytosensor), ouabain and its 1,5,19-phosphate at 10(-5) M reduced the rate of extracellular acidification by 15-20%. During inhibitor washout, the rate of recovery from the 1,5,19-phosphate analogue was approximately 3 times faster than ouabain. The 1,11,19-phosphate at 10(-4) M elicited a weak ( approximately 7%) response, and the effects reversed approximately 44-fold faster than ouabain. Studies with purified Na(+),K(+)-ATPase showed that ouabain and its 1,5,19-phosphate analogue were of similar efficacy (EC(50) = 1.1 and 5.2 x 10(-7) M, respectively) and >100-fold more potent than the 1,11,19-phosphate analogue. Studies of the binding kinetics showed that the 1,5,19-phosphate analogue bound 3-fold and dissociated 16-fold faster from the purified Na(+),K(+)-ATPase than ouabain. Both analogues were competitive inhibitors of 3H-ouabain binding. Taken together, these results suggest that the marked conformational flexibility of the A-ring in ouabain ordinarily slows the initial binding of this steroid to the sodium pump. However, once ouabain is bound, flection of the steroidal A- and BC-rings is critical for the maintenance of high-affinity binding. Our results indicate that the ouabain-binding site is comprised of structurally mobile elements and highlight the roles that synchronization between receptor and ligand dynamics play as determinants of biological activity in this system.