Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have identified and characterized cDNAs encoding a novel zebrafish Na,K-ATPase alpha subunit. The full-length cDNA encodes a 1023-amino-acid-long peptide which shows greatest homology to zebrafish alpha1 polypeptides. Radiation hybrid mapping localized the new gene (atp1a1a.5) to linkage group 1 in close proximity to the previously identified cluster of Na,K-ATPase alpha1 genes. The expression of atp1a1a.5 in zebrafish embryos was analyzed using whole-mount in situ hybridization. From mid-somitogenesis through 48 h post fertilization (hpf), atp1a1a.5 transcripts were detected in the pronephric duct, ear, and mucous cells. This expression pattern continues through 108 hpf, when high levels of expression were also detected in the intestinal bulb.
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PMID:Cloning, mapping, and developmental expression of a sixth zebrafish Na,K-ATPase alpha1 subunit gene (atp1a1a.5). 1451 87

The two cell types in the lens, epithelium and fiber, have a very different specific activity of Na,K-ATPase; activity is much higher in the epithelium. However, judged by Western blot, fibers and epithelium express a similar amount of both Na,K-ATPase alpha and beta subunit proteins. Na,K-ATPase protein abundance does not tally with Na,K-ATPase activity. Studies were conducted to examine whether protein synthesis plays a role in maintenance of the high Na,K-ATPase activity in lens epithelium. An increase of cytoplasmic sodium was found to increase Na,K-ATPase protein expression in the epithelium, but not in the fibers. The findings illustrate the ability of lens epithelium to synthesize new Na,K-ATPase protein as a way to boost Na,K-ATPase in response to cell damage or pathological events. Methionine incorporation studies suggested Na,K-ATPase synthesis may also play a role in day to day preservation of high Na,K-ATPase activity. Na,K-ATPase protein in lens epithelial cells appeared to be continually synthesized and degraded. Experiments with cycloheximide suggest that specific activity of Na,K-ATPase in the lens epithelium may depend on the ability of the cells to continuously synthesize fresh Na,K-ATPase proteins. However, other factors such as phosphorylation of Na,K-ATPase alpha subunit may also influence Na,K-ATPase activity. When intact lenses were exposed to the agonist thrombin, Na,K-ATPase activity was diminished, but the response was suppressed by inhibitors of the Src family of non-receptor tyrosine kinases. Thrombin elicited tyrosine phosphorylation of lens epithelium membrane proteins, including a 100 kDa protein band thought to be the Na,K-ATPase alpha 1 subunit. It remains to be determined whether a tyrosine phosphorylation mechanism contributes to the low activity of Na,K-ATPase in lens fibers.
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PMID:Regulation of Na,K-ATPase function in the lens. 1459 82

Several members of the FXYD protein family are tissue-specific regulators of Na,K-ATPase that produce distinct effects on its apparent K(+) and Na(+) affinity. Little is known about the interaction sites between the Na,K-ATPase alpha subunit and FXYD proteins that mediate the efficient association and/or the functional effects of FXYD proteins. In this study, we have analyzed the role of the transmembrane segment TM9 of the Na,K-ATPase alpha subunit in the structural and functional interaction with FXYD2, FXYD4, and FXYD7. Mutational analysis combined with expression in Xenopus oocytes reveals that Phe(956), Glu(960), Leu(964), and Phe(967) in TM9 of the Na,K-ATPase alpha subunit represent one face interacting with the three FXYD proteins. Leu(964) and Phe(967) contribute to the efficient association of FXYD proteins with the Na,K-ATPase alpha subunit, whereas Phe(956) and Glu(960) are essential for the transmission of the functional effect of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase. The relative contribution of Phe(956) and Glu(960) to the K(+) effect differs for different FXYD proteins, probably reflecting the intrinsic differences of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase. In contrast to the effect on the apparent K(+) affinity, Phe(956) and Glu(960) are not involved in the effect of FXYD2 and FXYD4 on the apparent Na(+) affinity of Na,K-ATPase. The mutational analysis is in good agreement with a docking model of the Na,K-ATPase/FXYD7 complex, which also predicts the importance of Phe(956), Glu(960), Leu(964), and Phe(967) in subunit interaction. In conclusion, by using mutational analysis and modeling, we show that TM9 of the Na,K-ATPase alpha subunit exposes one face of the helix that interacts with FXYD proteins and contributes to the stable interaction with FXYD proteins, as well as mediating the effect of FXYD proteins on the apparent K(+) affinity of Na,K-ATPase.
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PMID:Structural and functional interaction sites between Na,K-ATPase and FXYD proteins. 1523 69

It now generally is agreed that Na,K-ATPase, in addition to its role in the maintenance of Na+ and K+ gradients across the cell membrane, plays a role in communicating information from the extracellular environment to intracellular signaling pathways. It was reported recently that interaction between ouabain-bound Na,K-ATPase and the 1,4,5-trisphosphate receptor (IP3R) triggers slow calcium oscillations and activation of NF-kappaB. Here it is demonstrated that this signaling pathway can serve to prevent cell death and promote cell growth. Rat renal proximal tubular cells in primary culture first were grown in the presence of 10% serum and then exposed to 0.2% serum for 24 h to induce apoptosis. Serum starvation increased the apoptotic index from 1.21 +/- 0.26 to 14.01 +/- 1.17%. Ouabain in concentrations that did not inhibit Na,K-ATPase activity (1 to 10 nM) completely abolished the apoptotic effect of serum starvation. Ouabain protection from apoptosis was not observed when release of calcium from intracellular stores via the IP3R was prevented. It was shown that the NH2 terminal tail of the Na,K-ATPase alpha subunit plays a key role in ouabain-triggered calcium oscillations. It was found that ouabain did not protect from apoptosis in serum-deprived cells that expressed a mutant Na,K-ATPase alpha subunit with deletion of the NH2 terminal tail. Ouabain exposure (10 nM for 24 h) significantly increased translocation of NF-kappaB from cytoplasm to nucleus. Helenalin, an inhibitor of NF-kappaB, abolished the antiapoptotic effect of ouabain. Ouabain (0.1 to 10 nM) also was found to stimulate proliferation and increase the viability of kidney cells. These effects were abolished when release of calcium via the IP3R was prevented.
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PMID:Low doses of ouabain protect from serum deprivation-triggered apoptosis and stimulate kidney cell proliferation via activation of NF-kappaB. 1670 66

FXYD1 is a major regulatory subunit of the Na,K-ATPase and the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinases A and C in heart and skeletal muscle sarcolemma. It is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the enzyme complex in a tissue-specific and physiological-state-specific manner. Here, we present the three-dimensional structure of FXYD1 determined in micelles by NMR spectroscopy. Structure determination was made possible by measuring residual dipolar couplings in weakly oriented micelle samples of the protein. This allowed us to obtain the relative orientations of the helical segments and information about the protein dynamics. The structural analysis was further facilitated by the inclusion of distance restraints, obtained from paramagnetic spin label relaxation enhancements, and by refinement with a micelle depth restraint, derived from paramagnetic Mn line broadening effects. The structure of FXYD1 provides the foundation for understanding its intra-membrane association with the Na,K-ATPase alpha subunit and suggests a mechanism whereby the phosphorylation of conserved Ser residues, by protein kinases A and C, could induce a conformational change in the cytoplasmic domain of the protein to modulate its interaction with the alpha subunit.
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PMID:Structure of the Na,K-ATPase regulatory protein FXYD1 in micelles. 1751 73

We characterized a family consisting of four mammalian proteins of unknown function (NKAIN1, 2, 3 and 4) and a single Drosophila ortholog dNKAIN. Aside from highly conserved transmembrane domains, NKAIN proteins contain no characterized functional domains. Striking amino acid conservation in the first two transmembrane domains suggests that these proteins are likely to function within the membrane bilayer. NKAIN family members are neuronally expressed in multiple regions of the mouse brain, although their expression is not ubiquitous. We demonstrate that mouse NKAIN1 interacts with the beta1 subunit of the Na,K-ATPase, whereas Drosophila ortholog dNKAIN interacts with Nrv2.2, a Drosophila homolog of the Na,K-ATPase beta subunits. We also show that NKAIN1 can form a complex with another beta subunit-binding protein, MONaKA, when binding to the beta1 subunit of the Na,K-ATPase. Our results suggest that a complex between mammalian NKAIN1 and MONaKA is required for NKAIN function, which is carried out by a single protein, dNKAIN, in Drosophila. This hypothesis is supported by the fact that dNKAIN, but not NKAIN1, induces voltage-independent amiloride-insensitive Na(+)-specific conductance that can be blocked by lanthanum. Drosophila mutants with decreased dNKAIN expression due to a P-element insertion in the dNKAIN gene exhibit temperature-sensitive paralysis, a phenotype also caused by mutations in the Na,K-ATPase alpha subunit and several ion channels. The neuronal expression of NKAIN proteins, their membrane localization and the temperature-sensitive paralysis of NKAIN Drosophila mutants strongly suggest that this novel protein family may be critical for neuronal function.
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PMID:A novel family of transmembrane proteins interacting with beta subunits of the Na,K-ATPase. 1760 67

The FXYD membrane proteins constitute a family of conserved auxiliary subunits of the Na,K-ATPase, and have been the focus of recent attention due to their ability to finely regulate the activity of the enzyme complex in various physiological settings. In this review we describe the structures of the proteins, as well as their dynamics and their associations with the lipid bilayer membrane, which we have recently determined by NMR spectroscopy. Although the proteins are relatively small, their genes contain as many as six to nine small exons, and the coincidence of structured protein segments with their genetic elements suggests assembly from discrete structural modules through exon shuffling. The three-dimensional structures and backbone dynamics provide the foundation for understanding their intra-membrane association with the Na,K-ATPase alpha subunit, and the structure of FXYD1 suggests a mechanism whereby the phosphorylation of conserved Ser residues, by protein kinases A and C, could induce a conformational change in the cytoplasmic domain of the protein, to modulate its interaction with the alpha subunit.
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PMID:Structures of the FXYD regulatory proteins in lipid micelles and membranes. 1800 Jul 45

Alveolar hypoxia occurs during ascent to high altitude and is also observed in patients with ARDS and acute hypoxemic respiratory failure, in which alveolar flooding is associated with a decrease in edema fluid clearance and increased mortality. The mechanisms that lead to the impairment of alveolar fluid clearance are not completely understood. Alveolar fluid reabsorption is accomplished mostly by active Na+ transport across the alveolar epithelium which creates an osmotic gradient responsible for the clearance of lung edema from the alveolar spaces. In vivo and in vitro hypoxia inhibits both the epithelial sodium channels, responsible for the apical sodium entry, and the basolateral Na,K-ATPase, responsible for Na+ extrusion. We have shown that acute hypoxia inhibits Na,K-ATPase function by promoting its endocytosis from the plasma membrane to intracellular compartments. This process is mediated by the generation of mitochondrial reactive oxygen species (ROS) as shown by pharmacological and genetic approaches. Hypoxia and ROS promote the PKC-zeta dependent phosphorylation of the Na,K-ATPase alpha subunit triggering its endocytosis in a clathrin-AP2 dependent process. The phosphorylation occurs at the Ser-18 in the alpha subunit N-terminus, and mutation of this serine prevents both the decrease in function and the endocytosis. More prolonged hypoxia causes the ubiquitination and degradation of Na,K-ATPase. Thus, methods that counterbalance the inhibition of edema clearance during hypoxia and improve the lung's ability to clear pulmonary edema are needed. As such, a better understanding of the mechanisms that increase Na,K-ATPase function, (i.e., activation of dopaminergic or adrenergic receptors, gene transfer) may lead to the development of therapeutic approaches to upregulate the Na-K-ATPase function and increase edema clearance.
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PMID:Hypoxic inhibition of alveolar fluid reabsorption. 1826 95

Hypoxia promotes Na,K-ATPase endocytosis via protein kinase C zeta (PKC zeta)-mediated phosphorylation of the Na,K-ATPase alpha subunit. Here, we report that hypoxia leads to the phosphorylation of 5'-AMP-activated protein kinase (AMPK) at Thr172 in rat alveolar epithelial cells. The overexpression of a dominant-negative AMPK alpha subunit (AMPK-DN) construct prevented the hypoxia-induced endocytosis of Na,K-ATPase. The overexpression of the reactive oxygen species (ROS) scavenger catalase prevented hypoxia-induced AMPK activation. Moreover, hypoxia failed to activate AMPK in mitochondrion-deficient rho(0)-A549 cells, suggesting that mitochondrial ROS play an essential role in hypoxia-induced AMPK activation. Hypoxia-induced PKC zeta translocation to the plasma membrane and phosphorylation at Thr410 were prevented by the pharmacological inhibition of AMPK or by the overexpression of the AMPK-DN construct. We found that AMPK alpha phosphorylates PKC zeta on residue Thr410 within the PKC zeta activation loop. Importantly, the activation of AMPK alpha was necessary for hypoxia-induced AMPK-PKC zeta binding in alveolar epithelial cells. The overexpression of T410A mutant PKC zeta prevented hypoxia-induced Na,K-ATPase endocytosis, confirming that PKC zeta Thr410 phosphorylation is essential for this process. PKC zeta activation by AMPK is isoform specific, as small interfering RNA targeting the alpha1 but not the alpha2 catalytic subunit prevented PKC zeta activation. Accordingly, we provide the first evidence that hypoxia-generated mitochondrial ROS lead to the activation of the AMPK alpha1 isoform, which binds and directly phosphorylates PKC zeta at Thr410, thereby promoting Na,K-ATPase endocytosis.
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PMID:Alpha1-AMP-activated protein kinase regulates hypoxia-induced Na,K-ATPase endocytosis via direct phosphorylation of protein kinase C zeta. 1938 Apr 82

This study examined how the quaternary organic ammonium ion, benzyltriethylamine (BTEA), binds to the Na,K-ATPase to produce membrane potential (V(M))-dependent inhibition and tested the prediction that such a V(M)-dependent inhibitor would display electrogenic binding kinetics. BTEA competitively inhibited K(+) activation of Na,K-ATPase activity and steady-state (86)Rb(+) occlusion. The initial rate of (86)Rb(+) occlusion was decreased by BTEA to a similar degree whether it was added to the enzyme prior to or simultaneously with Rb(+), a demonstration that BTEA inhibits the Na,K-ATPase without being occluded. Several BTEA structural analogues reversibly inhibited Na,K-pump current, but none blocked current in a V(M)-dependent manner except BTEA and its para-nitro derivative, pNBTEA. Under conditions that promoted electroneutral K(+)-K(+) exchange by the Na,K-ATPase, step changes in V(M) elicited pNBTEA-activated ouabain-sensitive transient currents that had similarities to those produced with the K(+) congener, Tl(+). pNBTEA- and Tl(+)-dependent transient currents both displayed saturation of charge moved at extreme negative and positive V(M), equivalence of charge moved during and after step changes in V(M), and similar apparent valence. The rate constant (k(tot)) for Tl(+)-dependent transient current asymptotically approached a minimum value at positive V(M). In contrast, k(tot) for pNBTEA-dependent transient current was a "U"-shaped function of V(M) with a minimum value near 0 mV. Homology models of the Na,K-ATPase alpha subunit suggested that quaternary amines can bind to two extracellularly accessible sites, one of them located at K(+) binding sites positioned between transmembrane helices 4, 5, and 6. Altogether, these data revealed important information about electrogenic ion binding reactions of the Na,K-ATPase that are not directly measurable during ion transport by this enzyme.
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PMID:Quaternary benzyltriethylammonium ion binding to the Na,K-ATPase: a tool to investigate extracellular K+ binding reactions. 1962 94


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