Fine specificity mapping and topography of an isozyme-specific epitope of the Na,K-ATPase catalytic subunit

An isozyme-specific domain of the catalytic subunit of the Na,K-ATPase has been identified using a monoclonal antibody, McK1. The antibody's specificity was confirmed by its ability to stain proteolytic fingerprints of the Na,K-ATPase. The antibody recognized the alpha I isozyme of the rat Na,K-ATPase, but not the alpha II or alpha III isozymes. It recognized native and sodium dodecyl sulfate-denatured Na,K-ATPase and specifically stained basolateral membranes of the renal tubule. It bound to rat alpha I with highest affinity, but also cross-reacted with mouse, monkey, and human alpha I. It did not cross-react with sheep, pig, chicken, Torpedo, or dog alpha I. Fine specificity mapping was used to deduce the most likely antibody binding sites, based on comparison of eight amino acid sequences from cDNA clones. Two potential binding sites were found at widely separated locations. Limited tryptic digestion of the native enzyme was then used to demonstrate that the binding site was close to the N-terminal end of the Na,K-ATPase. The binding site is predicted to include the following essential amino acid sequence: Asp-Lys-Lys-Ser-Lys-Lys in rat alpha I or Asp-Lys-Lys-Gly-Lys-Lys in human alpha I. The antibody was found to bind to opened, but not to sealed right-side-out vesicles isolated from the rat renal medulla, demonstrating that the N-terminal end of the Na,K-ATPase is exposed at the interior of the cell.     

Neutral endopeptidase neprilysin is copurified with Na,K-ATPase from rabbit outer medulla and hydrolyzes its α-subunit

Preparations of Na,K-ATPase from outer medulla of rabbit kidney purified in accordance with the method of P. L. Jorgensen were shown to contain as admixture a protease that moves with α-subunit (∼100 kDa) as a single protein band during one-dimensional SDS-PAGE. The electro-elution of proteins of this band from polyacrylamide gel results in the appearance of two protein fragments (∼67 and 55 kDa) that are stained with polyclonal antibodies against Na,K-ATPase α-subunit. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis showed that the neutral membrane-bound endopeptidase neprilysin is located in one protein band together with the Na,K-ATPase α-subunit. Addition of thiorphan, a specific inhibitor of neutral endopeptidase, eliminates proteolysis of the α-subunit. The data demonstrate that Na,K-ATPase α-subunit may be a natural target for neprilysin.     

The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.     

Occlusion of Rb+ after extensive tryptic digestion of shark rectal gland Na,K-ATPase.

Na,K-ATPase from rectal glands of Squalus acanthias has been subjected to proteolysis with trypsin. The E1- and E2-forms of the enzyme can be distinguished from the inactivation patterns at low trypsin concentrations, as previously seen with kidney enzyme. Extensive degradation by trypsin in the presence of 5 mM Rb+ yields membrane fragments with a 19 kDa peptide as the major proteolytic fragment of the alpha-subunit. The sequence of the N-terminal 40 residues of this peptide is almost identical to that of a similar proteolytic fragment isolated by Capasso et al. (Capasso, J.M., Hoving, S., Tal, D.M., Goldshleger, R. and Karlish, S.J.D. (1992) J. Biol. Chem. 267, 1150-1158) using kidney Na,K-ATPase. Rb+ occlusion can be fully retained under these circumstances, supporting the findings with kidney enzyme that only minor parts of the alpha-subunit are required to form a functional occlusion-site.     

Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton

In striking contrast to most other transporting epithelia (e.g., urinary or digestive systems), where Na,K-ATPase is expressed basolaterally, the retinal pigment epithelium (RPE) cells display Na,K-ATPase pumps on the apical membrane. We report here studies aimed to identify the mechanisms underlying this polarity "reversal" of the RPE Na,K-ATPase. By immunofluorescence on thin frozen sections, both alpha and beta subunits were localized on the apical surface of both freshly isolated rat RPE monolayers and RPE monolayers grown in culture. The polarity of the RPE cell is not completely reversed, however, since aminopeptidase, an apically located protein in kidney epithelia, was also found on the apical surface of RPE cells. We used subunit- and isoform-specific cDNA probes to determine that RPE Na,K-ATPase has the same isoform (alpha 1) as the one found in kidney. Ankyrin and fodrin, proteins of the basolateral membrane cytoskeleton of kidney epithelial cells known to be associated with the Na,K-ATPase (Nelson, W. J., and R. W. Hammerton. 1989. J. Cell Biol. 110:349-357) also displayed a reversed apical localization in RPE and were intimately associated to Na,K-ATPase, as revealed by cross-linking experiments. These results indicate that an entire membrane-cytoskeleton complex is assembled with opposite polarity in RPE cells. We discuss our observations in the context of current knowledge on protein sorting mechanisms in epithelial cells.     

Two isozymes of the Na,K-ATPase have distinct antigenic determinants

Two isozymes of the Na,K-ATPase were purified from rat renal medulla and rat brainstem axolemma, and antisera were raised in rabbits. When antibody titers were measured, two sera showed specificity for either the kidney or axolemma Na,K-ATPases and had limited cross-reactivity which could be removed by cross-adsorption. In blots of polyacrylamide gels, these sera reacted with only the alpha or alpha (+) Na,K-ATPase catalytic subunits, while they cross-reacted with both types of beta subunits. Two other sera each recognized both alpha and alpha (+), indicating that the catalytic subunit isozymes have additional shared antigenic determinants. A comparison of the Na,K-ATPases from the brains of different vertebrate species indicates that birds and fish differ from mammals and amphibians in the manifestation of Na,K-ATPases isozymes. Neither neuraminidase nor endoglycosidase F treatment eliminated specific antibody reaction or affected the electrophoretic mobilities of the alpha and alpha (+) subunits, although endoglycosidase F increased the mobilities of the two types of beta subunits to similar final apparent molecular weights. Blots of the peptide fragments produced by incomplete papain and trypsin digests of the alpha and alpha (+) subunits were stained with the specific sera, and the patterns of immunoreactive fragments were found to be markedly different. The results suggest that the antigenic differences reside in differences in the primary protein sequences of the two isozymes.     

Induction of sodium pump beta 1-subunit mRNA expression during hepatocellular growth transitions in vitro and in vivo.

Previous suggestions (Hubert, J. J., Schenk, D. B., Skelly, H., and Leffert, H. L. (1986) Biochemistry 25, 4156-4163) of tissue-specific isoforms or nonexistence of hepatic Na,K-ATPase beta 1-subunits were reevaluated by quantifying beta 1-subunit mRNA levels in quiescent and proliferating liver. RNA was extracted from caudate liver lobes of sham or 67% hepatectomized adult rats and from primary cultures of adult rat hepatocytes that simulate developmental and regenerating growth transitions. Northern blot analysis with a 32P-labeled full-length Na,K-ATPase beta 1-cDNA probe (Mercer, R. W., Schneider, J. W., Savitz, A., Emmanuel, J., Benz, T.J., and Levenson, R. (1986) Mol. Cell. Biol. 6, 3884-3890) revealed four (approximately 2.7, 2.4, 1.7-1.8, and 1.5 kilobases) low abundance mRNA species in quiescent tissue, freshly isolated hepatocytes, and cultured hepatocytes derived from lag or late stationary phase (1-2 days or 11-12 days postplating, respectively). In contrast, proliferating liver from 4 h post-67% hepatectomized rats or cultured hepatocytes in logarithmic growth phase contained levels of beta 1-subunit mRNA which exceeded quiescent levels by 4-35-fold. Membrane Na,K-ATPase activity also increased 2-3-fold during liver regeneration 12-24 h after partial hepatectomy. When proliferation in vitro was augmented by transforming growth factor-alpha, a hepatocyte mitogen, or reinitiated in late stationary phase by a change to fresh culture medium containing rat serum, beta 1-subunit mRNA expression was restimulated 4-20-fold. Parallel measurements of alpha-tubulin mRNA induction showed relatively nonsynchronous or invariant changes during hepatocyte proliferative transitions; similar results were obtained after Northern blots with a sodium pump alpha I-subunit cDNA probe. No detectable hybridization signals were observed when either rat kidney or hepatocyte RNAs from freshly isolated and cultured cells or regenerating tissues were probed for the sodium pump 3.4-kilobase mRNA beta 2-isoform. These observations suggest that enhanced hepatic beta 1-subunit gene expression is linked specifically to growth-associated increases in Na,K-ATPase activity, hepatocyte proliferation, and mitogen activation.     

Identification of two isoforms of the catalytic subunit of Na,K-ATPase in myocytes from adult rat heart

The present study demonstrates that two forms of the alpha catalytic subunit of the Na,K-ATPase are present in rat heart and originate from cardiomyocytes. They were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction and alkylation of the sulfhydryl groups. The two forms were identified on immunoblots using two specific antisera against either the alpha subunit from Bufo marinus kidney and the alpha and beta subunits from lamb kidney. Comparison of the two forms to the alkylated Na,K-ATPase from rat kidney (containing one catalytic subunit) and from rat brain (containing alpha and alpha + subunits) suggested that, in rat cardiac myocytes, the form with a fast migration rate (alpha F) corresponds to the alpha subunit of low ouabain affinity and the one with a slow migration rate (alpha S), to a subunit of high ouabain affinity. Thus, the existence of two isoforms of the catalytic subunit in cardiac myocytes accounts well for the biphasic ouabain inhibition of the Na,K-ATPase activity and for the biphasic inotropic responsiveness to cardiac glycosides of the rat heart.     

Expression of the β-subunit isoforms of the Na,K-ATpase in rat embryo tissues,inner ear and choroid plexus

Summary— We report evidence of the apical localization of the two Na, K-ATPase β-subunit isoforms in cells of the inner ear and of the choroid plexus of the rat. To this end, we generated isoform-specific antisera against the human Na, K-ATPase β1 and β2 subunits. These polyclonal rabbit antisera were raised against truncated β-isoform proteins that were made in E coli with pET expression vectors. Deglycosylation of the native antigen with N-endoglycosidase F shows four bands in the β1 isoform and five bands in the β2 iso-form immunoblots. In E15 rat embryos, the β1 isoform was detected in brain, heart and kidney and the β2 isoform only in brain. While β-subunit mRNA expression (Watts AG, Sanchéz-Watts G, Emanuel JR, Levenson R 1991 Proc Natl Acad Sci USA 88, 7425–7429), and immunoblotting and enzymatic activity have been determined (Zlokovic BV, Mackic JB, Wang L, McComb JG, McDonough A 1993 J Biol Chem 268, 8019–8025), very little is known about the specific localization of each β-isoform in the epithelia of choroid plexus and inner ear. Immunocytochemical preparations of 15-day-old whole rat embryos and adult rat brain showed an enhanced staining for the β1 and β2 isoforms in the apical membrane of the ampullary crests of the inner ear's semicircular ducts and in the cuboidal cells of the choroid plexus     

Porcine kidney extract contains a specific inhibitor of the ouabain-sensitive alpha2-isoform of Na, K-ATPase present in rat diaphragm fibres

In experiments on isolated rat diaphragm muscle, acetylcholine (100 nmol/l) hyperpolarized muscle fibres due to activation of the alpha 2 isoform of Na,K-ATPase. This hyperpolarization was blocked in a dose-dependent manner by ouabain (K0.5 = 8 +/- 4 nmol/l) as well as by a solution of porcine kidney extract (10 kDa cut-off filtration), with the K0.5 approximately equal to a 1:20,000-fold dilution. The inhibitory activity of the developed slowly over a period of 3 hours and, in contrast to ouabain, was still present after 1 hour of washing. Ouabain, but not the extract, inhibits Rb+ uptake in human erythrocytes that only express the alpha = 1 isoform of Na, K-ATPase. Our data suggest that in rat skeletal muscle the alpha 1 isoform of Na,K-ATPase is primarily responsible for ionic homeostasis, while the alpha 2 isoform provides a "regulatable" function and may be controlled by cholinergic stimulation and/or endogenous digitalis-like factors (EDLFs). Porcine kidney extract contains a factor (M. W. < 10 kDa) that selectively inhibits the rat alpha 2 isoform and differs from ouabain. Our experimental protocol can be used as a highly sensitive physiological assay for factors that selectively inhibit the alpha 2 isoform of Na,K-ATPase.     

Isoform-specific effects of charged residues at borders of the M1-M2 loop of the Na,K-ATPase alpha subunit

The Na,K-ATPase is specifically inhibited by the cardiac glycoside, ouabain. Via a largely undefined mechanism, the ouabain affinity of the Na,K-ATPase can be manipulated by mutating the residues at the borders of the first extracellular (M1-M2) loop of the alpha subunit [Price, E. M., Rice, D. A., and Lingrel, J. B. (1990) J. Biol. Chem. 265, 6638-6641]. To address this issue, we compared the effects of two combinations of charged residues at the M1-M2 loop border, R113, D124 and D113,R124 (numbered according to the rat alpha1 subunit), on the ouabain sensitivity of the alpha1 and alpha2 isoforms. We report that ouabain sensitivity is dependent not only upon the identity of the residues at the M1-M2 loop border but also upon the context into which they are introduced. Furthermore, at low concentrations of ATP, the identity of the residues at the M1-M2 loop border affects the regulation of ATP hydrolysis by potassium in an isoform-specific manner. Analysis of chimeric alpha subunits reveals that the effects of potassium are determined primarily by the interaction of the N-terminus and M1-M2 loop with the C-terminal third of the alpha subunit. M1-M2 loop border residues may, therefore, influence ouabain sensitivity indirectly by altering the stability or structure of the intermediate of the Na,K-ATPase catalytic cycle which is competent to bind ouabain.     

Na,K-ATPase in dog red cells. Immunological identification and maturation-associated degradation by the proteolytic system

The Na,K-ATPase of red cells from high K+ and low K+ dogs was studied immunologically by using antibodies raised against dog kidney enzyme. Anti-alpha subunit IgGs, which also recognized alpha (+) from brain enzyme, identified the larger subunit of erythrocyte Na,K-ATPase as a homogeneous polypeptide with Mr = 96,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting. In addition, erythrocyte Na,K-ATPase, purified by immunoaffinity chromatography on a monoclonal antibody-coupled column, showed the identity of its polypeptide composition to that of the renal enzyme. Furthermore, it was shown that reticulocyte lysates from high K+ and low K+ dogs substantially degraded 125I-Bolton-Hunter reagent-labeled Na,K-ATPase. This degradation of the enzyme protein was significantly enhanced by the addition of ATP and Mg2+. These results indicate that dog reticulocytes possess some mechanism for protein breakdown involving an ATP-dependent proteolytic system, resulting in the dramatic breakdown of Na,K-ATPase activity during dog reticulocyte maturation into erythrocytes (Maede, Y., and Inaba, M. (1985) J. Biol. Chem. 260, 3337-3343).     

Comparison of the substrate dependence properties of the rat Na,K-ATPase alpha 1, alpha 2, and alpha 3 isoforms expressed in HeLa cells.

The role of multiple isoforms for the alpha subunit of Na,K-ATPase is essentially unknown. To examine the functional properties of the three alpha subunit isoforms, we developed a system for the heterologous expression of Na,K-ATPase in which the enzymatic activity of each isoform can be independently analyzed. Ouabain-resistant forms of the rat alpha 2 and alpha 3 subunits were constructed by site-directed mutagenesis of amino acid residues at the extracellular borders of the first and second transmembrane domains (L111R and N122D for alpha 2 and Q108R and N119D for alpha 3). cDNAs encoding the rat alpha 1 subunit, which is naturally ouabain-resistant, and rat alpha 2 and alpha 3, which were mutated to ouabain resistance (designated rat alpha 2* and rat alpha 3*, respectively) were cloned into an expression vector and transfected into HeLa cells. Resistant clones were isolated and analyzed for ouabain-inhibitable ATPase activity in the presence of 1 microM ouabain, which inhibits the endogenous Na,K-ATPase present in HeLa cells (I50 approximately equal to 10 nM). The remaining activity corresponds to Na,K-ATPase molecules containing the transfected rat alpha 1, rat alpha 2*, or rat alpha 3* isoforms. Utilizing this system, we examined Na+, K+, and ATP dependence of enzyme activity. Na,K-ATPase molecules containing rat alpha 1 and rat alpha 2* exhibited a 2-3-fold higher apparent affinity for Na+ than those containing rat alpha 3* (apparent KNa+ (millimolar): rat alpha 1 = 1.15 +/- 0.13; rat alpha 2* = 1.05 +/- 0.11; rat alpha 3* = 3.08 +/- 0.06). Additionally, rat alpha 3* had a slightly higher apparent affinity for ATP (in the millimolar concentration range) compared with rat alpha 1 or rat alpha 2* (apparent K0.5 (millimolar): rat alpha 1 = 0.43 +/- 0.12; rat alpha 2* = 0.54 +/- 0.15; rat alpha 3* = 0.21 +/- 0.04) and all three isoforms has similar apparent affinities for K+ (apparent KK+: rat alpha 1 = 0.45 +/- 0.01; rat alpha 2* = 0.43 +/- 0.004; rat alpha 3* = 0.27 +/- 0.01). This study represents the first comparison of the functional properties of the three Na,K-ATPase alpha isoforms expressed in the same cell type.     

Separate Na,K-ATPase genes are required for otolith formation and semicircular canal development in zebrafish

We have investigated the role of Na,K-ATPase genes in zebrafish ear development. Six Na,K-ATPase genes are differentially expressed in the developing zebrafish inner ear. Antisense morpholino knockdown of Na,K-ATPase alpha1a.1 expression blocked formation of otoliths. This effect was phenocopied by treatment of embryos with ouabain, an inhibitor of Na,K-ATPase activity. The otolith defect produced by morpholinos was rescued by microinjection of zebrafish alpha1a.1 or rat alpha1 mRNA, while the ouabain-induced defect was rescued by expression of ouabain-resistant zebrafish alpha1a.1 or rat alpha1 mRNA. Knockdown of a second zebrafish alpha subunit, alpha1a.2, disrupted development of the semicircular canals. Knockdown of Na,K-ATPase beta2b expression also caused an otolith defect, suggesting that the beta2b subunit partners with the alpha1a.1 subunit to form a Na,K-ATPase required for otolith formation. These results reveal novel roles for Na,K-ATPase genes in vestibular system development and indicate that different isoforms play distinct functional roles in formation of inner ear structures. Our results highlight zebrafish gene knockdown-mRNA rescue as an approach that can be used to dissect the functional properties of zebrafish and mammalian Na,K-ATPase genes.     

Co-localization and polarized distribution of Na,K-ATPase alpha 3 and beta 2 subunits in photoreceptor cells.

Na,K-ATPase plays a central role in the visual sensitivity of photoreceptors by driving the dark current of vision. The alpha 3 and beta 2 isoforms of Na,K-ATPase were previously shown to be the major alpha and beta subunit mRNAs expressed in photoreceptors. Here we compared the distribution of beta-subunits of the enzyme in the retina and kidney, using electron microscopic immunocytochemistry with specific antibodies against alpha 3, beta 1, and beta 2 isoforms as well as with an antibody (Ax2) that binds to alpha 2 and/or alpha 3 isoforms. Both the alpha 3 and beta 2 isoforms were localized to photoreceptor inner segments at highest labeling density between the base of the connecting cilium and the outer limiting membrane (OLM). Quantitative analysis of Ax2 antibody binding to alpha 3 revealed a significant decrease in labeling density below the OLM and above the base of the connecting cilium. Although the beta 2-subunit has been reported to have adhesive functions in glial cells in cerebellum, we detected beta 2 in the photoreceptor, a cell of neural origin, but not in the Mueller cell, the glial cell of the retina. Moreover, anti-beta 2 antibodies bound maximally to portions of photoreceptor cells not involved in cell-cell contact.     

Mechanistic basis for kinetic differences between the rat alpha 1, alpha 2, and alpha 3 isoforms of the Na,K-ATPase.

Previous studies showed that the alpha 1, alpha 2, and alpha 3 isoforms of the catalytic subunit of the Na,K-ATPase differ in their apparent affinities for the ligands ATP, Na(+), and K(+). For the rat isoforms transfected into HeLa cells, K'(ATP) for ATP binding at its low affinity site is lower for alpha 2 and alpha 3 compared with alpha 1; relative to alpha 1 and alpha 2, alpha 3 has a higher K'(Na) and lower K'(K) (Jewell, E. A., and Lingrel, J. B (1991) J. Biol. Chem. 266, 16925--16930; Munzer, J. S., Daly, S. E., Jewell-Motz, E. A., Lingrel, J. B, and Blostein, R. (1994) J. Biol. Chem. 269, 16668--16676). The experiments described in the present study provide insight into the mechanistic basis for these differences. The results show that alpha 2 differs from alpha1 primarily by a shift in the E(1) E(2) equilibrium in favor of E(1) form(s) as evidenced by (i) a approximately 20-fold increase in IC(50) for vanadate, (ii) decreased catalytic turnover, and (iii) notable stability of Na,K-ATPase activity at acidic pH. In contrast, despite its lower K'(ATP) compared with alpha 1, the E(1) E(2) poise of alpha 3 is not shifted toward E(1). Distinct intrinsic interactions with Na(+) ions are underscored by the marked selectivity for Na(+) over Li(+) of alpha 3 compared with either alpha1 or alpha 2 and higher K'(Na) for cytoplasmic Na(+), which persists over a 100-fold range in proton concentration, independent of the presence of K(+). The kinetic analysis also suggests alpha 3-specific differences in relative rates of partial reactions, which impact this isoform's distinct apparent affinities for both Na(+) and K(+).     

Thyroid hormone regulates alpha and alpha + isoforms of Na,K-ATPase during development in neonatal rat brain

The brain contains two molecular forms of Na,K-ATPase designated alpha found in non-neuronal cells and neuronal soma and alpha + found in axolemma. Previously we have shown that the abundance of both forms (determined by immunoblots) as well as Na,K-ATPase activity increases 10-fold between 4 days before and 20 days after birth (Schmitt, C. A., and McDonough, A. A. (1986) J. Biol. Chem. 261, 10439-10444). Hypothyroidism in neonates blunts these increases. Neonatal, but not adult brain Na,K-ATPase is thyroid hormone (triiodothyronine, T3) responsive. This study defines the period during which brain Na,K-ATPase responds to T3. The start of the critical period was defined by comparing Na,K-ATPase activity and alpha and alpha + abundance in hypothyroid and euthyroid neonates (birth to 30 days of age). For all parameters, euthyroid was significantly higher by 15 days of age. The end of the critical period was defined by dosing hypothyroid neonates with T3 daily (0.1 micrograms/g body weight) beginning at increasing days of age, and sacrificing all at 30 days then assaying enzyme activity and abundance. Those starting T3 treatment on or before day 19 were restored to euthyroid levels of Na,K-ATPase activity and abundance, while those starting T3 treatment on or after day 22 remained at hypothyroid levels of enzyme activity and abundance. We conclude that brain Na,K-ATPase alpha and alpha + isoforms are sensitive to T3 by as late as 15 days of age and that the period of thyroid hormone responsiveness is over by 22 days.     

Participation of Na,K-ATPase in FGF-2 secretion: rescue of ouabain-inhibitable FGF-2 secretion by ouabain-resistant Na,K-ATPase alpha subunits

We have examined the relationship between Na,K-ATPase and FGF-2 secretion in transfected primate cells. FGF-2 lacks a classic hydrophobic export signal, and the mechanisms mediating its secretion are unknown. To monitor secretion, a FLAG epitope tag was inserted into the carboxyl terminus of the 18 kDa form of human FGF-2, and the construct was transfected into either human HEK 293 or monkey CV-1 cells. Exported FGF-2 was detected in the culture medium using the FLAG-specific monoclonal antibody M2. FGF-2 secretion from HEK 293 or CV-1 cells was linear over time and sensitive to inhibition by the cardiac glycoside ouabain, a specific inhibitor of the Na,K-ATPase. In contrast, the secretion of FGF-8 (an FGF family member that contains a hydrophobic secretory signal) was not inhibited by treatment of HEK 293 or CV-1 cells with ouabain. FGF-2 secretion was also assayed in CV-1 cells expressing the naturally ouabain-resistant rodent Na,K-ATPase alpha1 subunit. In cells expressing the rodent alpha1 subunit, FGF-2 secretion was unaffected by high levels of ouabain, indicating that the rodent alpha1 subunit was capable of rescuing ouabain-inhibitable FGF-2 export. Expression of ouabain-resistant mutants of the rodent alpha2 and alpha3 subunits, or the naturally ouabain-resistant rodent alpha4 subunit, also supported FGF-2 secretion in ouabain-treated cells. Taken together, our studies are consistent with the idea that the Na,K-ATPase plays a prominent role in regulating FGF-2 secretion, although none of the alpha subunit isoforms exhibited specificity with regard to FGF-2 export.     

A new variant of the gamma subunit of renal Na,K-ATPase. Identification by mass spectrometry, antibody binding, and expression in cultured cells

The gamma subunit is a specific regulator of Na,K-ATPase expressed mainly in kidney. On SDS-polyacryylamide gel electrophoresis, gamma runs as a doublet, but the origin and significance of the doublet is obscure. Mass spectrometry of the gamma chains of rat kidney Na, K-ATPase shows that gamma(a) (upper) has a mass of 7184.0 +/- 1 Da (carbamidomethyl cysteine), corresponding closely to that for the published sequence without the initiator methionine, while gamma(b) (lower) has a mass of 7337.9 +/- 1Da. Tryptic peptide mapping and sequencing by mass spectrometry reveals that the seven N-terminal residues of gamma(a), TELSANH, are replaced by Ac-MDRWYL in gamma(b), but otherwise the chains are identical. Antibodies raised against peptides TELSANHC and MDRWYLC recognize either gamma(a) or gamma(b) of the Na,K-ATPase, respectively. gamma(a) or gamma(b) cDNAs have been expressed in human embryonic kidney and HeLa cells. The major bands expressed correspond to gamma(a) or gamma(b) of renal Na, K-ATPase. Additional minor bands seen after transfection, namely gamma(a)' in human embryonic kidney and gamma(b)' in HeLa, are presumably cell-specific modifications. The present work clarifies earlier uncertainty regarding doublets seen in kidney and in transfected cells. In particular, the results show that renal Na, K-ATPase contains two variants of the gamma subunit with different sequences but otherwise are unmodified. We discuss the possible functional significance of the two variants.