Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump.

Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+ and K+ transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369 to allow the transfer of ATP's terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ-phosphate group of ATP to the Asp369 is achieved, analogous molecular modeling of the M4-M5 loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain. However, binding of Mg2+•ATP to any of these sites in the "open conformation" may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between "open conformation" <==> "semi-open conformation <==> "closed conformation" in the absence of 2Mg2+•ATP. The cytoplasmic loop's conformational change to the "semi-open conformation"-characterized by a hydrogen bond between Arg543 and Asp611-triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the "closed conformation" the phosphorylation of Asp369 in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis.

Endogenous and exogenous cardiac glycosides and their mechanisms of action.

Cardiac glycosides have been used for decades to treat congestive heart failure. The recent identification of cardiotonic steroids such as ouabain, digoxin, marinobufagenin, and telocinobufagin in blood plasma, adrenal glands, and hypothalamus of mammals led to exciting new perspectives in the pathology of heart failure and arterial hypertension. Biosynthesis of ouabain and digoxin occurs in adrenal glands and is under the control of angiotensin II, endothelin, and epinephrine released from cells of the midbrain upon stimulation of brain areas sensing cerebrospinal Na(+) concentration and, apparently, the body's K(+) content. Rapid changes of endogenous ouabain upon physical exercise may favor the economy of the heart by a rise of intracellular Ca(2)(+) levels in cardiac and atrial muscle cells. According to the sodium pump lag hypothesis, this may be accomplished by partial inhibition of the sodium pump and Ca(2+) influx via the Na(+)/Ca(2+) exchanger working in reverse mode or via activation of the Na(+)/K(+)-ATPase signalosome complex, generating intracellular calcium oscillations, reactive oxygen species, and gene activation via nuclear factor-kappaB or extracellular signal-regulated kinases 1 and 2. Elevated concentrations of endogenous ouabain and marinobufagenin in the subnanomolar concentration range were found to stimulate proliferation and differentiation of cardiac and smooth muscle cells. They may have a primary role in the development of cardiac dysfunction and failure because (i) offspring of hypertensive patients evidently inherit elevated plasma concentrations of endogenous ouabain; (ii) such elevated concentrations correlate positively with cardiac dysfunction, hypertrophy, and arterial hypertension; (iii) about 40% of Europeans with uncomplicated essential hypertension show increased concentrations of endogenous ouabain associated with reduced heart rate and cardiac hypertrophy; (iv) in patients with advanced arterial hypertension, circulating levels of endogenous ouabain correlate with BP and total peripheral resistance; (v) among patients with idiopathic dilated cardiomyopathy, high circulating levels of endogenous ouabain and marinobufagenin identify those individuals who are predisposed to progressing more rapidly to heart failure, suggesting that endogenous ouabain (and marinobufagenin) may contribute to toxicity upon digoxin therapy. In contrast to endogenous ouabain, endogenous marinobufagenin may act as a natriuretic substance as well. It shows a higher affinity for the ouabain-insensitive alpha(1) isoform of Na(+)/K(+)-ATPase of rat kidney tubular cells and its levels are increased in volume expansion and pre-eclampsia. Digoxin, which is synthesized in adrenal glands, seems to counteract the hypertensinogenic action of ouabain in rats, as do antibodies against ouabain, for example, (Digibind) and rostafuroxin (PST 2238), a selective ouabain antagonist. It lowers BP in ouabain- and adducin-dependent hypertension in rats and is a promising new class of antihypertensive medication in humans.

Endogenous and exogenous cardiac glycosides: their roles in hypertension, salt metabolism, and cell growth.

Cardiotonic steroids (CTS), long used to treat heart failure, are endogenously produced in mammals. Among them are the hydrophilic cardenolide ouabain and the more hydrophobic cardenolide digoxin, as well as the bufadienolides marinobufagenin and telecinobufagin. The physiological effects of endogenous ouabain on blood pressure and cardiac activity are consistent with the "Na(+)-lag" hypothesis. This hypothesis assumes that, in cardiac and arterial myocytes, a CTS-induced local increase of Na(+) concentration due to inhibition of Na(+)/K(+)-ATPase leads to an increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) via a backward-running Na(+)/Ca(2+) exchanger. The increase in [Ca(2+)](i) then activates muscle contraction. The Na(+)-lag hypothesis may best explain short-term and inotropic actions of CTS. Yet all data on the CTS-induced alteration of gene expression are consistent with another hypothesis, based on the Na(+)/K(+)-ATPase "signalosome," that describes the interaction of cardiac glycosides with the Na(+) pump as machinery activating various signaling pathways via intramembrane and cytosolic protein-protein interactions. These pathways, which may be activated simultaneously or selectively, elevate [Ca(2+)](i), activate Src and the ERK1/2 kinase pathways, and activate phosphoinositide 3-kinase and protein kinase B (Akt), NF-kappaB, and reactive oxygen species. A recent development indicates that new pharmaceuticals with antihypertensive and anticancer activities may be found among CTS and their derivatives: the antihypertensive rostafuroxin suppresses Na(+) resorption and the Src-epidermal growth factor receptor-ERK pathway in kidney tubule cells. It may be the parent compound of a new principle of antihypertensive therapy. Bufalin and oleandrin or the cardenolide analog UNBS-1450 block tumor cell proliferation and induce apoptosis at low concentrations in tumors with constitutive activation of NF-kappaB.

Endogenous cardiac glycosides: hormones using the sodium pump as signal transducer.

The search for an endogenous digitalis has led to the identification of the cardenolides ouabain and digoxin and the bufadienolide marinobufagenin in mammalian tissues and biological fluids. Ouabain's release from adrenal glands is under the control of epinephrine and angiotensin II; hence, its blood concentration changes rapidly on physical exercise. It also is controlled by brain areas sensing cerebrospinal Na+ concentration and apparently the body's K+ content because urinary K+ loss leads to an increase in its plasma concentration as well. Long-term treatment of rats with ouabain results in arterial hypertension, and 50% of Caucasians with low-renin hypertension have increased plasma concentrations of this cardenolide. Levels of digoxin, which is synthesized from acetate in adrenal glands, increase slightly in blood on prolonged exercise. It counteracts the hypertensinogenic action of ouabain in rats, as does the ouabain antagonist PST 2238. The plasma concentration of the bufadienolide marinobufagenin is increased after cardiac infarction. It may show natriuretic properties because it inhibits the alpha1 isoform of Na+/K+-adenosine triphosphatase (ATPase), the main sodium pump isoform of the kidney, much better than other sodium pump isoforms. These effects of endogenous cardiac glycosides are observed at concentrations that do not inhibit the sodium pump. Apparently, Na+/K+-ATPase is used by these steroids as a signal transducer to activate tissue proliferation, heart contractility, arterial hypertension, and natriuresis via various intracellular signaling pathways.

Ouabain-like compound changes rapidly on physical exercise in humans and dogs: effects of beta-blockade and angiotensin-converting enzyme inhibition.

Ouabain, an inhibitor of the sodium pump, has been identified as a constituent of bovine adrenal glands. We were interested whether the release of this cardiotonic steroid is stimulated by physical exercise. Hence, athletes and healthy dogs were subjected to ergometry. Ouabain-like compound (OLC) was measured in venous blood by enzyme-linked immunosorbent assay as well as by (86)Rb+ uptake inhibition (as ouabain equivalents). OLC increased in venous blood of athletes after 15 minutes of ergometry from 2.5+/-0.5 to 86.0+/-27.2 nmol/L (n=51; P<0.001), as did the concentration of a circulating inhibitor of the sodium pump from 7.3+/-1.7 to 129.8+/-51 nmol/L (ouabain equivalents, P<0.05). Half-maximal increase in heart rate and systolic blood pressure occurred at 5.1+/-1.2 nmol/L and at 30+/-1 nmol/L OLC, respectively. On rest, OLC decreased in humans and dogs with a half-life of 3 to 5 minutes. In beagles exposed to moderate exercise on a treadmill for 13 minutes, levels of OLC increased 46-fold (from 3.7+/-0.8 to 166.9+/-91.8 nmol/L; n=6; P<0.005). This effect was suppressed when the dogs had been treated for 3 weeks with the beta1-adrenergic receptor blocker atenolol or the angiotensin-converting enzyme inhibitor benazepril. We conclude that OLC changes rapidly during exercise and is under the control of norepinephrine and angiotensin II.

The phosphatase activity of the isolated H4-H5 loop of Na+/K+ ATPase resides outside its ATP binding site.

The structural stability of the large cytoplasmic domain (H(4)-H(5) loop) of mouse alpha(1) subunit of Na(+)/K(+) ATPase (L354-I777), the number and the location of its binding sites for 2'-3'-O-(trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) and p-nitrophenylphosphate (pNPP) were investigated. C- and N-terminal shortening revealed that neither part of the phosphorylation (P)-domain are necessary for TNP-ATP binding. There is no indication of a second ATP site on the P-domain of the isolated loop, even though others reported previously of its existence by TNP-N(3)ADP affinity labeling of the full enzyme. Fluorescein isothiocyanate (FITC)-anisotropy measurements reveal a considerable stability of the nucleotide (N)-domain suggesting that it may not undergo a substantial conformational change upon ATP binding. The FITC modified loop showed only slightly diminished phosphatase activity, most likely due to a pNPP site on the N-domain around N398 whose mutation to D reduced the phosphatase activity by 50%. The amino acids forming this pNPP site (M384, L414, W411, S400, S408) are conserved in the alpha(1-4) isoforms of Na(+)/K(+) ATPase, whereas N398 is only conserved in the vertebrates' alpha(1) subunit. The phosphatase activity of the isolated H(4)-H(5) loop was neither inhibited by ATP, nor affected by mutation of D369, which is phosphorylated in native Na(+)/K(+) ATPase.

The hydrogen bonds between Arg423 and Glu472 and other key residues, Asp443, Ser477, and Pro489, are responsible for the formation and a different positioning of TNP-ATP and ATP within the nucleotide-binding site of Na(+)/K(+)-ATPase.

Mutation of Arg(423) at the N-domain of Na(+)/K(+)-ATPase resulted in a large decrease of both TNP-ATP and ATP binding. Thus, this residue, localized outside the binding pocket, seems to play a key role in supporting the proper structure and shape of the binding site. In addition, mutation of Glu(472) also caused a large decrease of both TNP-ATP and ATP binding. On the basis of our computer model, we hypothesized that a hydrogen bond between Arg(423) and Glu(472) supports the connection of two opposite halves of the ATP-binding pocket. To verify this hypothesis, we have also prepared the construct containing both these mutations. Binding of neither TNP-ATP nor ATP to this double mutant differed from binding to any of the single mutants. This strongly supported the existence of the hydrogen bond between Arg(423) and Glu(472). Similarly, the conserved residue Pro(489) seems to be substantial for the proper interaction of the third and fourth beta-strands of the N-domain, which both contain residues that take part in ATP binding. Mutation of Asp(443) affected only ATP, but not TNP-ATP, binding, suggesting that these ligands adopt different positions in the nucleotide-binding pocket. On the basis of a recently published crystal structure [Håkansson, K. O. (2003) J. Mol. Biol. 332, 1175-1182], we improved our model and computed the interaction of these two ligands with the N-domain. This model is in good agreement with all previously reported spectroscopic data and revealed that Asp(443) forms a hydrogen bond with the NH(2) group of the adenosine moiety of ATP, but not TNP-ATP.

Glutamate receptors communicate with Na+/K+-ATPase in rat cerebellum granule cells: demonstration of differences in the action of several metabotropic and ionotropic glutamate agonists on intracellular reactive oxygen species and the sodium pump.

Two glutamate receptor agonists, NMDA (N-methyl-D-aspartic acid) and ACPD (cis-(1S/3R)-1-aminocyclopentane- 1,3-dicarboxylic acid), induce the reactive oxygen species (ROS) production in rat cerebellum granule cells, whereas the third one, 3-HPG (3-hydroxyphenylglycine), decreases this parameter. The simultaneous presence of 3-HPG, together with NMDA or ACPD, prevents the generation of ROS by neuronal cells. A similar effect of these ligands on Na+/K+-ATPase can be demonstrated: NMDA and ACPD inhibited the enzyme activity, but 3-HPG activated Na+/K+-ATPase and prevented its inhibition by NMDA or ACPD. In terms of current classification, NMDA is an agonist of ionotropic glutamate receptors of the so-called NMDA class, whereas ACPD and 3-HPG belong to metabotropic agonists, the former primarily being an activator of metabotropic glutamate receptors (mGluRs) of groups 2 and 3, and the latter, that of mGluRs of groups 1 and 5. Thus, the data presented illustrate the existence of diverse mechanisms of the cross talk between Na+/K+-ATPase and different glutamate receptors, as well as that between glutamate receptors of different classes.

Eight amino acids form the ATP recognition site of Na(+)/K(+)-ATPase.

Point mutations of a part of the H(4)-H(5) loop (Leu(354)-Ile(604)) of Na(+)/K(+)-ATPase have been used to study the ATP and TNP-ATP binding affinities. Besides the previously reported amino acid residues Lys(480), Lys(501), Gly(502), and Cys(549), we have found four more amino acid residues, viz., Glu(446), Phe(475), Gln(482), and Phe(548), completing the ATP-binding pocket of Na(+)/K(+)-ATPase. Moreover, mutation of Arg(423) has also resulted in a large decrease in the extent of ATP binding. This residue, localized outside the binding pocket, seems to play a key role in supporting the proper structure and shape of the binding site, probably due to formation of a hydrogen bond with Glu(472). On the other hand, only some minor effects were caused by mutations of Ile(417), Asn(422), Ser(445), and Glu(505).

Ouabain as a mammalian hormone.

Endogenous ouabain changes rapidly in humans and dogs upon physical exercise and is under the control of epinephrine and angiotensin II. Hence, the steroid acts as a rapidly acting hormone. A search for a specific binding globulin for cardiac glycosides in bovine plasma resulted in the identification of the d allotype of the micro chain of IgM whose hydrophobic surfaces interact with cardiotonic steroids and cholesterol. Such IgM complexes might be involved in the hepatic elimination of cardiotonic steroids. Thus, differences in the signaling cascade starting at Na(+),K(+)-ATPase must explain any differences in the action of ouabain and digoxin in the genesis of arterial hypertension.

Phe(475) and Glu(446) but not Ser(445) participate in ATP-binding to the alpha-subunit of Na(+)/K(+)-ATPase.

The ATP-binding site of Na(+)/K(+)-ATPase is localized on the large cytoplasmic loop of the alpha-subunit between transmembrane helices H(4) and H(5). Site-directed mutagenesis was performed to identify residues involved in ATP binding. On the basis of our recently developed model of this loop, Ser(445), Glu(446), and Phe(475) were proposed to be close to the binding pocket. Replacement of Phe(475) with Trp and Glu(446) with Gln profoundly reduced the binding of ATP, whereas the substitution of Ser(445) with Ala did not affect ATP binding. Fluorescence measurements of the fluorescent analog TNP-ATP, however, indicated that Ser(445) is close to the binding site, although it does not participate in binding.

Endogenous cardiac glycosides, a new class of steroid hormones.

The search for endogenous digitalis has led to the isolation of ouabain as well as several additional cardiotonic steroids of the cardenolide and bufadienolide type from blood, adrenals, and hypothalamus. The concentration of endogenous ouabain is elevated in blood upon increased Na(+) uptake, hypoxia, and physical exercise. Changes in blood levels of ouabain upon physical exercise occur rapidly. Adrenal cortical cells in tissue culture release ouabain upon addition of angiotensin II and epinephrine, and it is thought that ouabain is released from adrenal cortex in vivo. Ouabain levels in blood are elevated in 50% of Caucasians with low-renin hypertension. Infusion over several weeks of low concentrations of ouabain, but not of digoxin, induces hypertension in rats. A digoxin-like compound, which has been isolated from human urine and adrenals, as well various other endogenous cardiac glycosides may counterbalance their actions within a regulatory framework of water and salt metabolism. Marinobufagenin, for instance, whose concentration is increased after cardiac infarction, may show natriuretic properties because it inhibits the alpha1 isoform of Na(+)/K(+)-ATPase, the main sodium pump isoform of the kidney, much better than other sodium pump isoforms. In analogy to other steroid hormones, cardiotonic steroid hormones in blood are bound to a specific cardiac glycoside binding globulin. The discovery of ouabain as a new adrenal hormone affecting Na(+) metabolism and the development of the new ouabain antagonist PST 2238 allows for new possibilities for the therapy of hypertension and congestive heart failure. This will lead in turn to a better understanding of the disease on a physiological and endocrinological level and of the action of ouabain on the cellular level as a signal that is transduced to the plasma membrane as well as to the cell nucleus.