Modulation of Na,K-ATPase expression during early development of Xenopus laevis.

In amphibian and mammalian systems, regulation of Na+ transport via the Na,K-ATPase plays an important role in distinct developmental processes such as blastocoele formation and neurulation. In this study, we have followed the Na,K-ATPase activity, the biosynthesis, and the cellular accumulation of catalytic alpha-subunits after fertilization of Xenopus laevis eggs up to neurula formation. Our data show that Na,K-ATPase activity increases significantly between stages 4 and 6 and again between stages 13 and 24. The four-fold rise in Na,K-ATPase activity during blastocoele formation is not mediated by an increased cellular pool of alpha-subunits. On the other hand, a five-fold increase of the biosynthesis rate around midblastula precedes a progressive accumulation up to neurula stage mainly of alpha 1-subunits and to a lesser extent of a second alpha-immunoreactive species. In contrast, newly synthesized glycoproteinic beta 1-subunits of Na,K-ATPase cannot be detected up to late neurula. These data indicate that (1) upregulation of Na,K-ATPase activity during blastocoele and neurula formation are mediated by different regulation mechanisms and (2) alpha- and possibly beta-isoforms are expressed in a developmentally regulated fashion during early Xenopus development.     

Differential regulation of Na,K-ATPase alpha 1, alpha 2, and beta subunit mRNA and protein levels by thyroid hormone

The purpose of this study was to determine the effect of thyroid status on the Na,K-ATPase alpha isoforms and beta in rat heart, skeletal muscle, kidney, and brain at the levels of mRNA, protein abundance, and enzymatic activity. Northern and dot-blot analysis of RNA (euthyroid, hypothyroid, and triiodothyronine-injected hypothyroids = hyperthyroids) and immunoblot analysis of protein (euthyroid and hypothyroid) revealed isoform-specific regulation of Na,K-ATPase by thyroid status in kidney, heart, and skeletal muscle and no regulation of sodium pump subunit levels in the brain. In general, in the transition from euthyroid to hypothyroid alpha 1 mRNA and protein levels are unchanged in kidney and skeletal muscle and slightly decreased in heart, while alpha 2 mRNA and protein are decreased significantly in heart and skeletal muscle. In hypothyroid heart and skeletal muscle, the decrease in alpha 2 protein levels was much greater than the decrease in alpha 2 mRNA levels relative to euthyroid indicating translational or post-translational regulation of alpha 2 protein abundance by triiodothyronine status in these tissues. The regulation of beta subunit by thyroid status is tissue-dependent. In hypothyroid kidney beta mRNA levels do not change, but immunodetectable beta protein levels decrease relative to euthyroid, and the decrease parallels the decrease in Na,K-ATPase activity. In hypothyroid heart and skeletal muscle beta mRNA levels decrease; beta protein decreases in heart and was not detected in the skeletal muscle. These findings demonstrate that the euthyroid levels of expression of alpha 1 in heart, alpha 2 in heart and skeletal muscle, and beta in kidney, heart, and skeletal muscle are dependent on the presence of thyroid hormone.     

Covalent cross-links between the gamma subunit (FXYD2) and alpha and beta subunits of Na,K-ATPase: modeling the alpha-gamma interaction

This study describes specific intramolecular covalent cross-linking of the gamma to alpha and gamma to beta subunits of pig kidney Na,K-ATPase and rat gamma to alpha co-expressed in HeLa cells. For this purpose pig gammaa and gammab sequences were determined by cloning and mass spectrometry. Three bifunctional reagents were used: N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA), disuccinimidyl tartrate (DST), and 1-ethyl-3-[3dimethylaminopropyl]carbodiimide (EDC). NHS-ASA induced alpha-gamma, DST induced alpha-gamma and beta-gamma, and EDC induced primarily beta-gamma cross-links. Specific proteolytic and Fe(2+)-catalyzed cleavages located NHS-ASA- and DST-induced alpha-gamma cross-links on the cytoplasmic surface of the alpha subunit, downstream of His(283) and upstream of Val(440). Additional considerations indicated that the DST-induced and NHS-ASA-induced cross-links involve either Lys(347) or Lys(352) in the S4 stalk segment. Mutational analysis of the rat gamma subunit expressed in HeLa cells showed that the DST-induced cross-link involves Lys(55) and Lys(56) in the cytoplasmic segment. DST and EDC induced two beta-gamma cross-links, a major one at the extracellular surface within the segment Gly(143)-Ser(302) of the beta subunit and another within Ala(1)-Arg(142). Based on the cross-linking and other data on alpha-gamma proximities, we modeled interactions of the transmembrane alpha-helix and an unstructured cytoplasmic segment SKRLRCGGKKHR of gamma with a homology model of the pig alpha1 subunit. According to the model, the transmembrane segment fits in a groove between M2, M6, and M9, and the cytoplasmic segment interacts with loops L6/7 and L8/9 and stalk S5.     

Differential expression of the Na,K-ATPase alpha 1 and alpha 2 subunit genes in a murine myogenic cell line. Induction of the alpha 2 isozyme during myocyte differentiation

Expression of Na,K-ATPase catalytic alpha isoform (alpha 1, alpha 2, and alpha 3) and beta subunit genes in rodent muscle was investigated using the murine C2C12 myogenic cell line. RNA blot analyses of myoblasts revealed expression primarily of the alpha 1 mRNA and low levels of alpha 2 mRNA. Fusion of the proliferating myoblasts to form myotubes was accompanied by an approximate 12-fold induction of the alpha 2 mRNA. In contrast, expression of alpha 1 mRNA remained constant throughout myogenesis. The alpha 3 mRNA was not detected in either myoblasts or myotubes. The beta mRNA abundance also increased 2-3-fold during myotube formation. In rodent tissues, low and high affinity cardiac glycoside (e.g. ouabain) receptors have been shown to be associated with the Na,K-ATPase catalytic alpha 1 and alpha 2 isoform subunits, respectively. The existence of these two functional classes of Na,K-ATPase in myoblasts and myotubes correlated with the biphasic ouabain inhibition of Na,K-ATPase activity. Confluent myoblasts expressed primarily the alpha 1 isozyme (IC50 = 3.6 X 10(-5) M; 95% of total activity) and lesser amounts of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 5% of total activity). In contrast, the myotubes showed significant levels of the alpha 1 isozyme (IC50 = 4.0 X 10(-5) M; 68% of total activity) and, in addition, showed a 6-fold increase in the relative levels of the alpha 2 isozyme (IC50 = 1.1 X 10(-7) M; 32% of total activity). To quantitate further the expression of the high affinity, ouabain-sensitive alpha 2 isozyme, a whole cell [3H]ouabain-binding assay was used. Results revealed that myotubes have an approximately 6-fold greater concentration of [3H]ouabain-binding sites than myoblasts with an apparent dissociation constant (Kd) of 1.4 X 10(-7) M. The results indicate that muscle cells can express multiple isozymes of Na,K-ATPase and that expression of the alpha 2 isozyme is developmentally regulated during myogenesis.     

Cloning of the H,K-ATPase beta subunit. Tissue-specific expression, chromosomal assignment, and relationship to Na,K-ATPase beta subunits

We have isolated cDNA clones encoding the bovine and rat gastric H,K-ATPase beta subunit. A bovine abomasum lambda gt11 cDNA library was screened with a monoclonal antibody raised against the rabbit H,K-ATPase beta subunit. A single positive phage clone containing an approximately 900-base pair cDNA insert was identified as reactive with the antibody. The identity of the cDNA was established by comparing the deduced amino acid sequence with sequences of cyanogen bromide fragments of the porcine H,K-ATPase beta subunit. Polymerase chain reaction and rapid amplification of cDNA ends were used to generate a cDNA fragment encoding the carboxyl-terminal portion of the rat gastric H,K-ATPase beta subunit. A rat stomach cDNA library was screened with the polymerase chain reaction product, and several full-length beta subunit cDNA clones were identified. The open reading frame predicts a protein of 294 amino acids with a molecular weight of 33,689. The rat H,K-ATPase beta subunit shows 41% amino acid sequence identity to the rat Na,K-ATPase beta 2 subunit and shares a number of structural similarities with Na,K-ATPase beta subunit isoforms. By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the H,K-ATPase beta subunit gene to murine chromosome 8. Northern and Western blot analysis reveals that this gene is expressed exclusively in stomach. Our results suggest that the H,K-ATPase and Na,K-ATPase beta subunits evolved from a common ancestral gene and may play similar functional roles in enzyme activity.     

Functional role and immunocytochemical localization of the gamma a and gamma b forms of the Na,K-ATPase gamma subunit

The gamma 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 gamma is present primarily in the kidney as two main splice variants, gamma(a) and gamma(b), 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 gamma(a) and gamma(b). At least two singular effects are seen, irrespective of whether the variants are expressed in HeLa or rat alpha1-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-gamma) abrogate the first effect but not the second. In contrast, gamma(a) and gamma(b) show differences in their localization along the kidney tubule. Using anti-gamma (C-terminal) and antibodies to the rat alpha subunit as well as antibodies to identify cell types, double immunofluorescence showed gamma in the basolateral membrane of several tubular segments. Highest expression is in the medullary portion of the thick ascending limb (TAL), which contains both gamma(a) and gamma(b). In fact, TAL is the only positive tubular segment in the medulla. In the cortex, most tubules express gamma but at lower levels. Antibodies specific for gamma(a) and gamma(b) showed differences in their cortical location; gamma(a) is specific for cells in the macula densa and principal cells of the cortical collecting duct but not cortical TAL. In contrast, gamma(b) but not gamma(a) is present in the cortical TAL only. Thus, the importance of gamma(a) and gamma(b) may be related to their partially overlapping but distinct expression patterns and tissue-specific functions of the pump that these serve.     

Primary sequence and functional expression of a novel ouabain-resistant Na,K-ATPase. The beta subunit modulates potassium activation of the Na,K-pump.

In order to understand the molecular mechanism of ouabain resistance in the toad Bufo marinus, Na,K-ATPase alpha and beta subunits have been cloned and their functional properties tested in the Xenopus laevis oocyte expression system. According to sequence comparison between species, alpha 1, beta 1, and beta 3 isoforms were identified in a clonal toad urinary bladder cell line (TBM 18-23). The sequence of the alpha 1 isoform is characterized by two positively charged amino acids (Arg, Lys) at the N-terminal border of the H1-H2 extracellular loop and no charged amino acid at the C terminus, a pattern distinct from the ouabain-resistant rat alpha 1 isoform. The coexpression of alpha 1 beta 1 or alpha 1 beta 3 TBM subunits in the Xenopus oocyte resulted in the expression of identical maximum Na,K-pump currents with identical inhibition constant for ouabain (Ki) (alpha 1 beta 1: 53 +/- 3 microM; n = 7 vs. alpha 1 beta 3: 57 +/- 3.0 microM; n = 8) but distinct potassium half activation constant (K1/2) (alpha 1 beta 1: 0.87 +/- 0.08 mM, n = 16; alpha 1 beta 3: 1.29 +/- 0.07 mM, n = 17; p less than 0.005). We conclude that (i) the TBM alpha 1 isoform is necessary and sufficient to confer the ouabain resistant phenotype; (ii) the beta 3 or beta 1 subunit can associate with the alpha 1 equally well without affecting the ouabain-resistant phenotype; (iii) some specific sequence of the beta subunit can modulate the activation of the Na,K-pump by extracellular potassium ions.     

Antisera specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the Na,K-ATPase: differential expression of alpha and beta subunits in rat tissue membranes

We have developed a panel of antibodies specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the rat Na,K-ATPase. TrpE-alpha subunit isoform fusion proteins were used to generate three antisera, each of which reacted specifically with a distinct alpha subunit isotype. Western blot analysis of rat tissue microsomes revealed that alpha 1 subunits were expressed in all tissues while alpha 2 subunits were expressed in brain, heart, and lung. The alpha 3 subunit, a protein whose existence had been inferred from cDNA cloning, was expressed primarily in brain and copurified with ouabain-inhibitable Na,K-ATPase activity. An antiserum specific for the rat Na,K-ATPase beta subunit was generated from a TrpE-beta subunit fusion protein. Western blot analysis showed that beta subunits were present in kidney, brain, and heart. However, no beta subunits were detected in liver, lung, spleen, thymus, or lactating mammary gland. The distinct tissue distributions of alpha and beta subunits suggest that different members of the Na,K-ATPase family may have specialized functions.     

Structure of the alpha 1 subunit of horse Na,K-ATPase gene

Genomic DNA for Na,K-ATPase alpha 1 subunit was obtained from libraries of horse kidney genomic DNA in Charon 4A and in EMBL3 bacteriophages by screening with the full sized cDNA probe of the alpha 1 subunit of rat Na,K-ATPase as probe. The gene spans 30 kb and consists of 23 exons and 22 intervening sequences. Intron-exon boundaries were analyzed. The protein-coding nucleotide sequence encodes 1016 amino acids with an Mr of 112,264. The putative amino acid sequence of horse alpha 1 is 96-97% homologous to those of other mammalian species.     

Distinct regulatory effects of the Na,K-ATPase gamma subunit

The two variants of the gamma subunit of the rat renal sodium pump, gamma(a) and gamma(b), have similar effects on the Na,K-ATPase. Both increase the affinity for ATP due to a shift in the enzyme's E(1) <--> E(2) conformational equilibrium toward E(1). In addition, both increase K(+) antagonism of cytoplasmic Na(+) activation. To gain insight into the structural basis for these distinct effects, extramembranous N-terminal and C-terminal mutants of gamma were expressed in rat alpha1-transfected HeLa cells. At the N terminus, the variant-distinct region was deleted (gammaNDelta7) or replaced by alanine residues (gammaN7A). At the C terminus, four (gamma(a)CDelta4) or ten (gamma(a)CDelta10) residues were deleted. None of these mutations abrogates the K(+)/Na(+) antagonism as evidenced in a similar increase in K'(Na) seen at high (100 mm) K(+) concentration. In contrast, the C-terminal as well as N-terminal deletions (gammaNDelta7, gamma(a)CDelta4, and gamma(a)CDelta10) abolished the decrease in K'(ATP) seen with wild-type gamma(a) or gamma(b). It is concluded that different regions of the gamma chain mediate the distinct functional effects of gamma, and the effects can be long-range. In the transmembrane region, the impact of G41R replacement was analyzed since this mutation is associated with autosomal dominant renal Mg(2+)-wasting in man (Meij, I. C., Koenderink, J. B., van Bokhoven, H., Assink, K. F. H., Groenestege, W. T., de Pont, J. J. H. H. M., Bindels, R. J. M., Monnens, L. A. H., Van den Heuvel, L. P. W. J., and Knoers, N. V. A. M. (2000) Nat. Genet. 26, 265-266). The results show that Gly-41 --> Arg prevents trafficking of gamma but not alphabeta pumps to the cell surface and abrogates functional effects of gamma on alphabeta pumps. These findings underscore a potentially important role of gamma in affecting solute transport, in this instance Mg(2+) reabsorption, consequent to its primary effect on the sodium pump.     

Differential expression of the alpha 2 and beta messenger RNAs of Na,K-ATPase in developing brine shrimp as measured by in situ hybridization.

We used in situ hybridization histochemistry with synthetic oligonucleotide probes to localize the mRNAs encoding the alpha 2- and beta-mRNAs of Na,K-ATPase during development of the brine shrimp Artemia. The mRNAs of the alpha 2- and beta-subunit were of low abundance in the cysts; in addition, less mRNA of the beta-subunit was localized. During emergence (12 hr), there was an increase in alpha 2-subunit mRNA in the gut mucosa, but there was a burst in beta-subunit mRNA throughout. As development progressed, the mRNAs of both the alpha 2- and beta-subunits showed a distinct pattern of expression in which the mRNA in the salt gland was of greatest abundance, followed by epidermal cells and gut mucosa. After 36 hr the alpha 2-subunit mRNA began to decrease in all positive cells but still remained highest in the salt gland and the brain region, while the mRNA of the beta-subunit kept increasing in the gut mucosa. Finally, the greatest abundance of the beta-subunit mRNA shifted from the salt gland to the antenna gland and the epidermal cells in the tail region, but the alpha 2-subunit mRNA did not. The more widespread distribution of the beta-mRNA than alpha 2-mRNA at certain stages (e.g., there was no alpha 2-mRNA in the antenna gland at the adult stage) is in all likelihood due to the marked drop in the alpha 2-subunit and a rise in alpha 1-subunit previously seen by Peterson et al. on polyacrylamide gel electrophoresis, as development progresses.     

Oligomerization and maturation of Na,K-ATPase: functional interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha subunit

Subunit assembly plays an essential role in the maturation of oligomeric proteins. In this study, we have characterized the main structural and functional consequences of the assembly of alpha and beta subunits of Na,K-ATPase. Xenopus oocytes injected with alpha and/or beta cRNA were treated with brefeldin A, which permitted the accumulation of individual subunits or alpha-beta complexes in the ER. Only alpha subunits that are associated with beta subunits become resistant to trypsin digestion and cellular degradation. Similarly, assembly with beta subunits is necessary and probably sufficient for the catalytic alpha subunit to acquire its main functional properties at the level of the ER, namely the ability to adopt different ligand- dependent conformations and to hydrolyze ATP in an Na(+)- and K(+)- dependent, ouabain-inhibitable fashion. Not only the alpha but also the beta subunit undergoes a structural change after assembly, which results in a global increase in its protease resistance. Furthermore, extensive and controlled proteolysis assays on wild-type and NH2- terminally modified beta subunits revealed a K(+)-dependent interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha subunit, which is likely to be involved in the modulation of the K(+)- activation of the Na,K-pump transport activity. Thus, we conclude that the ER assembly process not only establishes the basic structural interactions between individual subunits, which are required for the maturation of oligomeric proteins, but also distinct, functional interactions, which are involved in the regulation of functional properties of mature proteins.     

The alpha and beta subunits of lamb kidney Na,K-ATPase are both glycoproteins

We have determined the carbohydrate compositions of the protein components of lamb kidney Na,K-ATPase. The α subunit contains a total of about 16 monosaccharide residues per mol of protein, while the β subunit contains about 36 residues per mol. The γ protein, a proteolipid associated with the Na,K-ATPase, contains only traces of carbohydrate. A comparison of our results with those of others shows considerable variability in the carbohydrate compositions of α and β subunits from different species.     

Na,K-ATPase alpha3 subunit in the goldfish retina during optic nerve regeneration

The goldfish optic nerve can regenerate after injury. To understand the molecular mechanism of optic nerve regrowth, we identified genes whose expression is specifically up-regulated during the early stage of optic nerve regeneration. A cDNA library constructed from goldfish retina 5 days after transection was screened by differential hybridization with cDNA probes derived from axotomized or normal retina. Of six cDNA clones isolated, one clone was identified as the Na,K-ATPase catalytic subunit alpha3 isoform by high- sequence homology. In northern hybridization, the expression level of the mRNA was significantly increased at 2 days and peaked at 5-10 days, and then gradually decreased and returned to control level by 45 days after optic nerve transection. Both in situ hybridization and immunohistochemical staining have revealed the location of this transient retinal change after optic nerve transection. The increased expression was observed only in the ganglion cell layer and optic nerve fiber layer at 5-20 days after optic nerve transection. In an explant culture system, neurite outgrowth from the retina 7 days after optic nerve transection was spontaneously promoted. A low concentration of ouabain (50-100 nm ) completely blocked the spontaneous neurite outgrowth from the lesioned retina. Together, these data indicate that up-regulation of the Na,K-ATPase alpha3 subunit is involved in the regrowth of ganglion cell axons after axotomy.     

Stress-induced expression of the gamma subunit (FXYD2) modulates Na,K-ATPase activity and cell growth

In kidney, the Na,K-ATPase is associated with a single span protein, the gamma subunit (FXYD2). Two splice variants are differentially expressed along the nephron and have a differential influence on Na,K-ATPase when stably expressed in mammalian cells in culture. Here we used a combination of gene induction and gene silencing techniques to test the functional impact of gamma by means other than transfection. NRK-52E cells (of proximal tubule origin) do not express gamma as a protein under regular tissue culture conditions. However, when they were exposed to hyperosmotic medium, induction of only the gammaa splice variant was observed, which was accompanied by a reduction in the rate of cell division. Kinetic analysis of stable enzyme properties from control (alpha1beta1) and hypertonicity-treated cultures (alpha1beta1gammaa) revealed a significant reduction (up to 60%) of Na,K-ATPase activity measured under V(max) conditions with little or no change in the amounts of alpha1beta1. This effect as well as the reduction in cell growth rate was practically abolished when gamma expression was knocked down using specific small interfering RNA duplexes. Surprisingly, a similar induction of endogenous gammaa because of hypertonicity was seen in rat cell lines of other than renal origin: C6 (glioma), PC12 (pheochromocytoma), and L6 (myoblasts). Furthermore, exposure of NRK-52E cells to other stress inducers such as heat shock, exogenous oxidation, and chemical stress also resulted in a selective induction of gammaa. Taken together, the data imply that induction of gammaa may have adaptive value by being a part of a general cellular response to genotoxic stress.     

Aldosterone modulates sodium kinetics of Na,K-ATPase containing an alpha 1 subunit in A6 kidney cell epithelia.

Short-term aldosterone (10(-6) M, 2.5 h) induces in A6-C1 cell epithelia an increase in Na transport, which is due to the in situ activation of the apical Na channel and, presumably, the basolateral Na pump (Na,K-ATPase). We have now directly measured the effect of aldosterone on the transport activity of endogenous Na pumps and hybrid Na pumps containing an exogenous alpha 1 subunit by measuring the pump current (Ip) across epithelia apically permeabilized with amphotericin B. Aldosterone (2.5 h) had no significant early effect on the maximal Ip, nor on the Na concentration required for half-maximal activation. In contrast, it increased the Ip at physiological intracellular Na concentrations (1.7-fold at 5 mM Na). This effect was blocked by the protein synthesis inhibitor cycloheximide. Hybrid pumps containing the transfected cardiotonic steroid-resistant alpha 1 subunit of Bufo marinus were also stimulated by aldosterone (2.5 h). A long aldosterone treatment (4 days) increased the maximal Ip produced by the endogenous pumps 1.5 to 2.1-fold. In conclusion, aldosterone acts on Na pumps containing an alpha 1 subunit in two ways. During its early phase of action it stimulates their transport activity by increasing their apparent Na affinity at physiological intracellular Na concentrations. In the long term it produces an increase in the maximal transport capacity, which corresponds to the known increase in the number of Na pumps.