The alpha-subunit of the colonic H+,K+-ATPase assembles with beta1-Na+,K+-ATPase in kidney and distal colon

Previous experiments from our laboratory (Codina, J., Kone, B. C., Delmas-Mata, J. T., and DuBose, T. D., Jr. (1996) J. Biol. Chem. 271, 29759-29763) demonstrated that the alpha-subunit of the colonic H+, K+-ATPase (HKalpha2) requires coexpression with a beta-subunit to support H+/K+ transport in a heterologous expression system (Xenopus laevis oocytes). In these studies, HKalpha2 formed stable and functional alpha.beta complexes when coexpressed with either the rat beta1-subunit of the Na+,K+-ATPase or the beta-subunit of the gastric H+,K+-ATPase, suggesting that different beta-subunits may interact with HKalpha2. The present studies tested this hypothesis by development and application of a specific antibody against HKalpha2 peptide. Subsequently, immunoprecipitation experiments were performed to determine if HKalpha2 co-precipitates with the same beta-subunit in organs known to express HKalpha2 protein. The data demonstrate that HKalpha2 assembles with beta1-Na+,K+-ATPase in the renal medulla and in distal colon.     

Constitutive activation of gastric H+,K+-ATPase by a single mutation

In the reaction cycle of P-type ATPases, an acid-stable phosphorylated intermediate is formed which is present in an intracellularly located domain of the membrane-bound enzymes. In some of these ATPases, such as Na+,K+-ATPase and gastric H+, K+-ATPase, extracellular K+ ions stimulate the rate of dephosphorylation of this phosphorylated intermediate and so stimulate the ATPase activity. The mechanism by which extracellular K+ ions stimulate the dephosphorylation process is unresolved. Here we show that three mutants of gastric H+,K+-ATPase lacking a negative charge on residue 820, located in transmembrane segment six of the alpha-subunit, have a high SCH 28080-sensitive, but K+-insensitive ATPase activity. This high activity is caused by an increased 'spontaneous' rate of dephosphorylation of the phosphorylated intermediate. A mutant with an aspartic acid instead of a glutamic acid residue in position 820 showed hardly any ATPase activity in the absence of K+, but K+ ions stimulated ATPase activity and the dephosphorylation process. These findings indicate that the negative charge normally present on residue 820 inhibits the dephosphorylation process. K+ ions do not stimulate dephosphorylation of the phosphorylated intermediate directly, but act by neutralizing the inhibitory effect of a negative charge in the membrane.     

Monoclonal antibodies to the alpha-subunit of the putative human H+,K+-ATPase encoded by the atp1al1 gene

The N-terminal fragment of ATP1AL1, the possible catalytic subunit of human ouabain-sensitive H+,K(+)-ATPase, was expressed in Escherichia coli cells as two recombinant proteins: the Ser14-Ile104 fragment or the same fragment containing His6 sequence at its N-end. The second protein was purified by metal-affinity chromatography and used as an antigen to construct two hybridoma lines producing antibodies of the IgM class. These monoclonal antibodies were shown to recognize not only the starting antigen but also the full-size recombinant ATP1AL1 protein and do not react with Na+,K(+)-ATPase.     

Phosphorylation of the catalyic alpha-subunit constitutes a triggering signal for Na+,K+-ATPase endocytosis

Inhibition of Na+,K+-ATPase activity by dopamine is an important mechanism by which renal tubules modulate urine sodium excretion during a high salt diet. However, the molecular mechanisms of this regulation are not clearly understood. Inhibition of Na+,K+-ATPase activity in response to dopamine is associated with endocytosis of its alpha- and beta-subunits, an effect that is protein kinase C-dependent. In this study we used isolated proximal tubule cells and a cell line derived from opossum kidney and demonstrate that dopamine-induced endocytosis of Na+,K+-ATPase and inhibition of its activity were accompanied by phosphorylation of the alpha-subunit. Inhibition of both the enzyme activity and its phosphorylation were blocked by the protein kinase C inhibitor bisindolylmaleimide. The early time dependence of these processes suggests a causal link between phosphorylation and inhibition of enzyme activity. However, after 10 min of dopamine incubation, the alpha-subunit was no longer phosphorylated, whereas enzyme activity remained inhibited due to its removal from the plasma membrane. Dephosphorylation occurred in the late endosomal compartment. To further examine whether phosphorylation was a prerequisite for subunit endocytosis, we used the opossum kidney cell line transfected with the rodent alpha-subunit cDNA. Treatment of this cell line with dopamine resulted in phosphorylation and endocytosis of the alpha-subunit with a concomitant decrease in Na+,K+-ATPase activity. In contrast, none of these effects were observed in cells transfected with the rodent alpha-subunit that lacks the putative protein kinase C-phosphorylation sites (Ser11 and Ser18). Our results support the hypothesis that protein kinase C-dependent phosphorylation of the alpha-subunit is essential for Na+,K+-ATPase endocytosis and that both events are responsible for the decreased enzyme activity in response to dopamine.     

A Glu329-->Gln variant of the alpha-subunit of the rat kidney Na+,K(+)-ATPase can sustain active transport of Na+ and K+ and Na+,K(+)-activated ATP hydrolysis with normal turnover number

An allelic variant of the ouabain-insensitive rat kidney Na+,K(+)-ATPase alpha 1-isoform was identified by chance in a cDNA library. The variant differed from the wild-type rat kidney Na+,K(+)-ATPase by a single G-to-C base substitution in the cDNA, which on amino acid level gave rise to a glutamine in place of the glutamate residue Glu329 previously suggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS-1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentration of ouabain highly cytotoxic to the parental cells containing only the endogenous COS-1 cell Na+,K(+)-ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain-insensitive Glu329-->Gln variant, whose cDNA was shown by polymerase chain reaction amplification to be stably integrated into the COS-1 cell genome. The maximum specific ATP hydrolysis activity of isolated plasma membranes of the Glu329-->Gln variant did not differ significantly from that of plasma membranes containing the wild type. A method was established for measurement of the phosphorylation capacity of the expressed Glu329-->Gln variant and wild-type enzyme, and it was thereby demonstrated that the variant had a turnover number similar if not identical to that of the wild-type.     

Overexpression of the Na+,K+-ATPase alpha1 subunit increases Na+,K+-ATPase function in A549 cells

Ron (the receptor for Macrophage Stimulating Protein) has never been implicated before in human malignancies or in cell transformation. In this report we show that Ron can acquire oncogenic potential by means of two amino acid substitutions-D1232V and M1254T-affecting highly conserved residues in the tyrosine kinase domain. The same mutations in Kit and Ret have been found associated with two human malignancies, mastocytosis and Multiple Endocrine Neoplasia type 2B (MEN2B), respectively. Both mutations caused Ron-mediated transformation of 3T3 fibroblasts and tumour formation in nude mice. Moreover, cells transformed by the oncogenic Ron mutants displayed high metastatic potential. The Ron mutant receptors were constitutively active and the catalytic efficiency of the mutated kinase was higher than that of wild-type Ron. Oncogenic Ron mutants enhanced activation of the Ras/MAPK cascade with respect to wild type Ron, without affecting the JNK/SAPK pathway. Expression of Ron mutants in 3T3 fibroblasts led to different patterns of tyrosine-phos-phorylated proteins. These data show that point mutations altering catalytic properties and possibly substrate specificity of the Ron kinase may force cells toward tumorigenesis and metastasis.     

Glucose decreases Na+,K+-ATPase activity in pancreatic beta-cells. An effect mediated via Ca2+-independent phospholipase A2 and protein kinase C-dependent phosphorylation of the alpha-subunit

In the pancreatic beta-cell, glucose-induced membrane depolarization promotes opening of voltage-gated L-type Ca2+ channels, an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i), and exocytosis of insulin. Inhibition of Na+,K+-ATPase activity by ouabain leads to beta-cell membrane depolarization and Ca2+ influx. Because glucose-induced beta-cell membrane depolarization cannot be attributed solely to closure of ATP-regulated K+ channels, we investigated whether glucose regulates other transport proteins, such as the Na+,K+-ATPase. Glucose inhibited Na+,K+-ATPase activity in single pancreatic islets and intact beta-cells. This effect was reversible and required glucose metabolism. The inhibitory action of glucose was blocked by pretreatment of the islets with a selective inhibitor of a Ca2+-independent phospholipase A2. Arachidonic acid, the hydrolytic product of this phospholipase A2, also inhibited Na+, K+-ATPase activity. This effect, like that of glucose, was blocked by nordihydroguaiaretic acid, a selective inhibitor of the lipooxygenase metabolic pathway, but not by inhibitors of the cyclooxygenase or cytochrome P450-monooxygenase pathways. The lipooxygenase product 12(S)-HETE (12-S-hydroxyeicosatetranoic acid) inhibited Na+,K+-ATPase activity, and this effect, as well as that of glucose, was blocked by bisindolylmaleimide, a specific protein kinase C inhibitor. Moreover, glucose increased the state of alpha-subunit phosphorylation by a protein kinase C-dependent process. These results demonstrate that glucose inhibits Na+, K+-ATPase activity in beta-cells by activating a distinct intracellular signaling network. Inhibition of Na+,K+-ATPase activity may thus be part of the mechanisms whereby glucose promotes membrane depolarization, an increase in [Ca2+]i, and thereby insulin secretion in the pancreatic beta-cell.     

Fourier transform infrared spectroscopy study of the secondary structure of the gastric H+,K+-ATPase and of its membrane-associated proteolytic peptides

Membrane topology of the H+,K+-ATPase has been studied after proteolytic degradation of the protein by proteinase K. Proteinase K had access to either the cytoplasmic part of the protein or to both sides of the membrane. Fourier transform infrared attenuated total reflection spectroscopy indicated that membrane-associated domain of the protein represented about 55% of the native protein, meanwhile the cytoplasmic part represented only 27% of the protein. The secondary structure of the ATPase and of its membrane-associated domains was investigated by infrared spectroscopy. The secondary structure of the membrane-associated structures and of the entire protein was quite similar (alpha-helices, 35%; beta-sheets, 35%; turns, 20%; random, 15%). These data were in agreement with 10 alpha-helical transmembrane segments but suggested a participation of beta-sheet structures in the membrane-associated part of the protein. Polarized infrared spectroscopy indicated that the alpha-helices were oriented nearly perpendicular to the membrane plane. No preferential orientation could be attributed to the beta-sheets. Monitoring the amide hydrogen/deuterium exchange kinetics demonstrated that the membrane associated part of the ATPase molecule is characterized by a relatively high accessibility to the solvent, quite different from that observed for bacteriorhodopsin membrane segments.     

Dephosphorylation kinetics of pig kidney Na+,K+-ATPase

The kinetics of K+-stimulated dephosphorylation of the Na+,K+-ATPase were investigated at pH 7.4, 24 degrees C, and an ATP concentration of 1.0 mM via the stopped-flow technique using the fluorescent label RH421. Two different mixing procedures were used: (a) premixing with ATP to allow phosphorylation to go to completion, followed by mixing with KCl; and (b) simultaneous mixing with ATP and KCl. Using mixing procedure (a), the dephosphorylation rate constant of enzyme complexed with K+ ions could be determined directly to be 190 s-1).     

Stimulation of ouabain-sensitive 86Rb+ uptake and Na+,K+-ATPase alpha-subunit phosphorylation by a cAMP-dependent signalling pathway in intact cells from rat kidney cortex

We investigated in intact cortical kidney tubules the role of PKA-mediated phosphorylation in the short-term control of Na+,K+-ATPase activity. The phosphorylation level of Na+,K+-ATPase was evaluated after immunoprecipitation of the enzyme from 32P-labelled cortical tubules and the cation transport activity of Na+,K+-ATPase was measured by ouabain-sensitive 86Rb+ uptake. Incubation of cells with cAMP analogues (8-bromo-cAMP, dibutyryl-cAMP) or with forskolin plus 3-isobutyl-1-methylxanthine increased the phosphorylation level of the Na+,K+-ATPase alpha-subunit and stimulated ouabain-sensitive 86Rb+ uptake. Inhibition of PKA by H-89 blocked the effects of dibutyryl-cAMP on both phosphorylation and 86Rb+ uptake processes. The results suggest that phosphorylation by PKA stimulates the Na+,K+-ATPase activity.     

A missense mutation of the gene for Na+,K(+)-ATPase alpha-subunit causes abnormal feeding behavior in Caenorhabditis elegans

The Na+,K(+)-ATPase activity of membranes from a behavioral mutant of C. elegans was found to be about one-third that of the wild-type. The levels of mRNA and polypeptide of Na+,K(+)-ATPase alpha-subunit in the mutant were as high as those in the wild-type, but the level of the phosphorylated intermediate of the Na+,K(+)-ATPase in the mutant worm was 80% lower than that in the wild-type. A single predicted amino acid replacement (Leu to Phe) was found in a highly conserved region in the alpha-subunit that is involved in the formation of phosphorylated intermediate. The abnormal feeding behavior of the mutant worm may be attributed to this missense mutation.     

ATP and acetyl phosphate induces molecular events near the ATP binding site and the membrane domain of Na+,K+-ATPase. The tetrameric nature of the enzyme

The addition of ATP to Mg2+-Na+-bound-probe labeled Na+,K+-ATPase preparations containing approximately 0.5 mol of pyridoxal 5'-diphospho-5'-adenosine (AP2PL) probe at Lys-480 and approximately 0.9 mol of fluorescein 5'-isothiocyanate (FITC) probe at Lys-501 showed a decrease and an increase in the AP2PL fluorescence intensity with neither significant ATP-dependent phosphorylation nor FITC fluorescence change. The rate constants for the fluorescence change increased nearly linearly with increasing ATP concentrations. The substitution of AcP for ATP decreased the FITC fluorescence rather monophasically, 8.5/s, which was followed by the half-site phosphorylation with same amount of components with different rate constant, 7.2 and 4.6/s, followed by a much slower increase in the two components of AP2PL fluorescence, 1.4 and 0.2/s. The addition of Na+ with increasing concentrations of ATP to the K+-bound AP2PL-FITC enzymes induced accelerations in the decrease and an increase in the AP2PL fluorescence intensity with two different increases in the FITC fluorescence intensity, showing that the same concentration of ATP is capable of inducing four different fluorescence changes. The addition of ATP to the Mg2+-Na+-bound enzymes modified with N-[p-(2-benzimidazolyl)phenyl]-maleimide (BIPM) at Cys-964 and retaining full Na+,K+-ATPase activity induced two different increases in BIPM fluorescence intensity. Each rate constant for the BIPM fluorescence change versus concentrations of ATP gave two intersecting straight lines. These data and the stoichiometries of fluorescence probe bindings and ATP- and AcP-dependent phosphorylation provide strong support for the conclusion that the functional membrane-bound Na+,K+-ATPase is a tetramer.     

Functional analysis and tissue-specific expression of Drosophila Na+,K+-ATPase subunits

We have previously purified and characterized a nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana [nerve antigen (NRV)] and identified two separate genes coding for three different proteins. All three proteins share homology with the beta subunits of Na+,K+-ATPase from various other species. In this study we have isolated a new Drosophila Na+,K+-ATPase alpha subunit cDNA clone (PSalpha; GenBank accession no. AF044974) and demonstrate expression of functional Na+,K+-ATPase activity when PSalpha mRNA is coinjected into Xenopus oocytes along with any of the three different Nrv mRNAs. Western blotting, RNase protection assays, and immunocytochemical staining of adult fly sections indicate that NRV2 is expressed primarily in the nervous system. Staining is most intense in the brain and thoracic ganglia and is most likely associated with neuronal elements. NRV1 is more broadly expressed in muscle and excretory tissue and also shows diffuse distribution in the nervous system. Similar to other species, Drosophila expresses multiple isoforms of Na+,K+-ATPase subunits in a tissue- and cell type-specific pattern. It will now be possible to use the advantages of Drosophila molecular and classical genetics to investigate the phenotypic consequences of altering Na+,K+-ATPase expression in various cell and tissue types.     

Serum antibodies to H+,K+-ATPase, serum pepsinogen A and Helicobacter pylori in relation to gastric mucosa morphology in patients with low or low-normal concentrations of serum cobalamins

The objective of this study was to investigate the effect of treatment with recombinant human granulocyte-colony stimulating factor (rhG-CSF) on the neutrophil count and function of preterm neonates with documented sepsis. For this purpose 62 preterm neonates with proven sepsis and 19 healthy preterm ones were studied. Of the 62 patients, 27 septic neonates had an absolute neutrophil count (ANC) > 5000/mm3 (group A) and were scheduled not to receive rhG-CSF and 35/62 had an ANC < 5000/mm3 (n=35) and were randomly assigned either to receive rhG-CSF (group B) or not to receive it (group C). rhG-CSF (10 microg/kg) was administered for 3 consecutive days (0, 1, 2). The ANC, plasma levels of G-CSF (ELISA), neutrophil respiratory burst activity (NRBA) and neutrophil expression of CD11a, CD11b and CD11c (flow cytometry) were measured in all septic neonates on days 0 (onset of sepsis), 1, 3 and 5 and in the healthy neonates once within the first 2 days of life. We found that on day 0, G-CSF levels of all groups of septic neonates were significantly higher than those of the healthy ones. The highest levels were observed in group A. NRBA was diminished only in groups B and C and the expression of CD11a and CD11c was reduced in all groups of septic neonates. Administration of rhG-CSF resulted in a rapid and significant increase in ANC, NRBA and CD11a, CD11b and CD11c expression that persisted throughout the follow up. CONCLUSION; The administration of granulocyte colony stimulating factor to septic neonates significantly increases the absolute granulocyte count and enhances the neutrophil respiratory burst and beta2 integrin expression.     

Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase

The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase were investigated via the stopped-flow technique using the fluorescent labels RH421 and BIPM. After the enzyme is mixed with MgATP, both labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau1 approximately 180 s-1 (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3 --> E2P(Na+)3 + ADP). The rate of the phosphorylation reaction measured by the acid quenched-flow technique was 190 s-1 at 100 microM ATP, suggesting that phosphorylation controls the kinetics of the RH421 signal and that the conformational change is very fast (>/=600 s-1). The rate of the RH421 signal was optimal at pH 7.5. The Na+ concentration dependence of 1/tau1 showed half-saturation at a Na+ concentration of 8-10 mM with positive cooperativity involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high affinity ATP binding site determined from the ATP concentration dependence of 1/tau1 was 7.0 (+/-0.6) microM, while the apparent Kd for the low affinity site and the rate constant for the E2 to E1 conformational change evaluated in the absence of Mg2+ were 143 (+/-17) microM and 相似文献   

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.     

Binding of pyrene isothiocyanate to the E1ATP site makes the H4-H5 cytoplasmic loop of Na+/K+-ATPase rigid

1-Pyreneisothiocyanate was shown to be an inhibitor of Na+/K+-ATPase. Reverse-phase HPLC and activity studies indicated binding of 1-pyreneisothiocyanate at the H4-H5 loop of the alpha subunit and competition with the fluorescein 5'-isothiocyanate for the E1ATP site. While fluorescein 5'-isothiocyanate, the fluorescent ATP pseudo-analog, was shown to be immobilized at the E1ATP site, there was no possibility to draw any conclusion about the flexibility of the E1ATP site due to its short lifetime. Employing 1-pyreneisothiocyanate as a long-lived fluorophore and a label for the E1ATP site, we found that the ATP-binding site of Na+/K+-ATPase and, in fact, the whole large intracellularly exposed H4-H5 loop of the catalytic alpha subunit is rigid and rotationally immobilized. This has important consequences for the molecular mechanism of the transport function.     

Microenvironment of the high affinity ATP-binding site of Na+/K+-ATPase is slightly acidic

Fluorescein-5'-isothiocyanate (FITC) was used to study the high-affinity ATP-binding site of Na+/K+-ATPase. The molar ratio of specifically bound FITC per alpha-subunit of Na+/K+-ATPase was found to be 0.5 as followed from pretreatment experiments with another specific E1ATP-inhibitor Cr(H2O)4AdoPP[CH2]P. This indicated an existence of one high affinity ATP-binding site (E1ATP-binding site) in the native (alphabeta)2-diprotomer of Na+/K+-ATPase. Fluorescence dual-excitation ratio of specifically bound FITC revealed that at external pH 7.5, the pH value inside the E1ATP-binding site is 6.95 +/- 0.18. In addition, FITC fluorescence quenching by anti-fluorescein and by iodide choline indicated the limited access of water into the small pocket of the E1ATP-binding site.     

Ion-sensitive endogenous regulators of Na+,K+-ATPase obtained from epithelium of rat small intestine

From epithelial layer of rat intestinal were selected water soluble substances which influenced on Na+,K(+)-ATPase activity in 2 different ways: substances with molecular weight 220 Da, 400 Da were its activators, substance with weight 150 Da-inhibitor. Na+,K(+)-ATPase activators preincubated previously with Na+ acquire properties of inhibitors. Bivalent cations Ca, Mg remove this effect of Na-ions.