Functional characterization of a testes-specific alpha-subunit isoform of the sodium/potassium adenosinetriphosphatase.

Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) alpha and beta subunits have been identified in mammals. The association of the various alpha and beta polypeptides results in distinct Na,K-ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase alpha4 isoform in Sf-9 cells using recombinant baculoviruses. When alpha4 and the Na pump beta1 subunit are coexpressed in the cells, Na, K-ATPase activity is induced. This activity is reflected by a ouabain-sensitive hydrolysis of ATP, by a Na(+)-dependent, K(+)-sensitive, and ouabain-inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K(+). Furthermore, the activity of alpha4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase alpha4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the beta1 and beta3 subunits. In insect cells, the alpha4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the alpha4beta1 and alpha4beta3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na(+), K(+), ATP, and ouabain. The enzymatic properties of alpha4beta1 and alpha4beta3 are, however, distinct from the other Na pump isozymes. A Na, K-ATPase activity with similar properties as the alpha4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the alpha4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.     

Roles of the Na,K-ATPase alpha4 isoform and the Na+/H+ exchanger in sperm motility

The Na,K-ATPase generates electrochemical gradients that are used to drive the coupled transport of many ions and nutrients across the plasma membrane. The functional enzyme is comprised of an alpha and beta subunit and families of isoforms for both subunits exist. Recent studies in this laboratory have identified a biological role for the Na,K-ATPase alpha4 isoform in sperm motility. Here we further investigate the role of the Na,K-ATPase carrying the alpha4 isoform, showing again that ouabain eliminates sperm motility, and in addition, that nigericin, a H+/K+ ionophore, and monensin, a H+/Na+ ionophore, reinitiate motility. These data, along with the observation that the K+ ionophore valinomycin has no effect on the motility of ouabain-inhibited sperm, suggest that ouabain may change intracellular H+ levels in a manner that is incompatible with sperm motility. We have also localized NHE1 and NHE5, known regulators of intracellular H+ content, to the same region of the sperm as the Na,K-ATPase alpha4 isoform. These data highlight the important role of the Na,K-ATPase alpha4 isoform in regulating intracellular H(+) levels, and provide evidence suggesting the involvement of the Na+/H+ exchanger, which is critical for maintaining normal sperm motility.     

FXYD6 is a novel regulator of Na,K-ATPase expressed in the inner ear

The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endolymph ionic composition and the endocochlear potential. A key protein in the maintenance of the electrochemical composition of the endolymph is the Na,K-ATPase. In this study, we have looked for the presence in the rat inner ear of members of the FXYD protein family, recently identified as tissue-specific modulators of Na,K-ATPase. Only FXYD6 is detected at the protein level. FXYD6 is expressed in various epithelial cells bordering the endolymph space and in the auditory neurons. FXYD6 co-localizes with Na,K-ATPase in the stria vascularis and can be co-immunoprecipitated with Na,K-ATPase. After expression in Xenopus oocytes, FXYD6 associates with Na,K-ATPase alpha1-beta1 and alpha1-beta2 isozymes, which are preferentially expressed in different regions of the inner ear and also with gastric and non-gastric H,K-ATPases. The apparent K(+) and Na(+) affinities of alpha1-beta1 and alpha1-beta2 isozymes are different. Association of FXYD6 with Na,K-ATPase alpha1-beta1 isozymes slightly decreases their apparent K(+) affinity and significantly decreases their apparent Na(+) affinity. On the other hand, association with alpha1-beta2 isozymes increases their apparent K(+) and Na(+) affinity. The effects of FXYD6 on the apparent Na(+) affinity of Na,K-ATPase and the voltage dependence of its K(+) effect are distinct from other FXYD proteins. In conclusion, this study defines the last FXYD protein of unknown function as a modulator of Na,K-ATPase. Among FXYD protein, FXYD6 is unique in its expression in the inner ear, suggesting a role in endolymph composition.     

Structural basis for alpha1 versus alpha2 isoform-distinct behavior of the Na,K-ATPase

We showed earlier that the kinetic behavior of the alpha2 isoform of the Na,K-ATPase differs from the ubiquitous alpha1 isoform primarily by a shift in the steady-state E(1)/E(2) equilibrium of alpha2 in favor of E(1) form(s). The aim of the present study was to identify regions of the alpha chain that confer the alpha1/alpha2 distinct behavior using a mutagenesis and chimera approach. Criteria to assess shifts in conformational equilibrium included (i) K(+) sensitivity of Na-ATPase measured at micromolar ATP, under which condition E(2)(K(+)) --> E(1) + K(+) becomes rate-limiting, (ii) changes in K'(ATP) for low affinity ATP binding, (iii) vanadate sensitivity of Na,K-ATPase activity, and (iv) the rate of the partial reaction E(1)P --> E(2)P. We first confirmed that interactions between the cytoplasmic domains of alpha2 that modulate conformational shifts are fundamentally similar to those of alpha1, suggesting that the predilection of alpha2 for E(1) state(s) is due to differences in primary structure of the two isoforms. Kinetic behavior of the alpha1/alpha2 chimeras indicates that the difference in E(1)/E(2) poise of the two isoforms cannot be accounted for by their notably distinct N termini, but rather by the front segment extending from the cytoplasmic N terminus to the C-terminal end of the extracellular loop between transmembranes 3 and 4, with a lesser contribution of the alpha1/alpha2 divergent portion within the M4-M5 loop near the ATP binding domain. In addition, we show that the E(1) shift of alpha2 results primarily from differences in the conformational transition of the dephosphoenzyme, (E(2)(K(+)) --> E(1) + K(+)), rather than phosphoenzyme (E(1)P --> E(2)P).     

Transport and pharmacological properties of nine different human Na, K-ATPase isozymes

Na,K-ATPase plays a crucial role in cellular ion homeostasis and is the pharmacological receptor for digitalis in man. Nine different human Na,K-ATPase isozymes, composed of 3 alpha and beta isoforms, were expressed in Xenopus oocytes and were analyzed for their transport and pharmacological properties. According to ouabain binding and K(+)-activated pump current measurements, all human isozymes are functional but differ in their turnover rates depending on the alpha isoform. On the other hand, variations in external K(+) activation are determined by a cooperative interaction mechanism between alpha and beta isoforms with alpha2-beta2 complexes having the lowest apparent K(+) affinity. alpha Isoforms influence the apparent internal Na(+) affinity in the order alpha1 > alpha2 > alpha3 and the voltage dependence in the order alpha2 > alpha1 > alpha3. All human Na,K-ATPase isozymes have a similar, high affinity for ouabain. However, alpha2-beta isozymes exhibit more rapid ouabain association as well as dissociation rate constants than alpha1-beta and alpha3-beta isozymes. Finally, isoform-specific differences exist in the K(+)/ouabain antagonism which may protect alpha1 but not alpha2 or alpha3 from digitalis inhibition at physiological K(+) levels. In conclusion, our study reveals several new functional characteristics of human Na,K-ATPase isozymes which help to better understand their role in ion homeostasis in different tissues and in digitalis action and toxicity.     

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.     

The human Na,K-ATPase alpha 4 isoform is a ouabain-sensitive alpha isoform that is expressed in sperm

The Na,K-ATPase generates electrochemical gradients across the plasma membrane that are responsible for numerous cellular and physiological processes. The active Na,K-ATPase is minimally composed of an alpha and a beta subunit and families of isoforms for both subunits exist. Recent studies have identified a physiological role for the rat Na,K-ATPase alpha4 isoform in sperm motility. However, very little is known about the human Na,K-ATPase alpha4 isoform other than its genomic sequence and structure and its mRNA expression pattern. Here, the human alpha4 isoform of the Na,K-ATPase is cloned, expressed, and characterized. Full length cDNAs encoding the putative human alpha4 isoform of the Na,K-ATPase were identified from a number of ESTs and a protein product corresponding to this isoform was shown to be expressed from these cDNAs. The human Na,K-ATPase alpha4 isoform protein was found to be expressed in mature sperm in human testes sections and it is localized specifically to the principle piece of human sperm. In addition, the presence of the Na,K-ATPase alpha4 isoform is absent in immature testes however its expression appears coincident with sexual maturity. And finally, the human Na,K-ATPase alpha4 isoform was shown to be as sensitive to cardiac glycoside inhibition as the human Na,K-ATPase alpha1 isoform. Considering the important role of the rat Na,K-ATPase alpha4 isoform in rat sperm motility, the demonstration that the human alpha4 isoform is a sperm-specific protein localized to the flagellum suggests a role for the human Na,K-ATPase alpha4 isoform in human sperm physiology.     

Identification of the cofilin-binding sites in the large cytoplasmic domain of Na,K-ATPase

Na,K-ATPase, an alpha, beta heterodimer, is found in the plasma membrane of all animal cells. The alpha chain is believed to have 10 transmembrane regions and a large cytoplasmic domain between the 4th and 5th transmembrane regions (H4-H5). In our previous report, the large (3rd) cytoplasmic domains of the alpha1 and alpha2 isoform were found to interact with cofilin, an actin-modulating protein, by the yeast two-hybrid system. Here we show that cofilin interacts only with the 3rd cytoplasmic domain of the alpha2 subunit but not with the 2nd, 4th, and 5th cytoplasmic domains or the cytoplasmic region of the beta subunit of Na,K-ATPase. We also demonstrate that cofilin interacts with the large cytoplasmic domains of the alpha1, alpha2 and alpha3 isoforms of Na,K-ATPase, but not with those of glucose transporter 1, glucose transporter 4, cystic fibrosis transmembrane conductance regulator and plasma membrane Ca-ATPase. We introduced 10 mutations into the 3rd cytoplasmic domain of Na,K-ATPase to identify the binding sites with cofilin. Eight of these mutants were single amino acid substitutions (R417Q, K470Q, K654G, D672A, K691A, R700G, R700A and D710G) and two were double mutant (K654GR700G and K719AK720A). Analysis of the activity of the reporter gene of these mutants shows that residues D672 and R700 of the 3rd cytoplasmic domain of Na,K-ATPase are involved in the interaction with cofilin.     

Sperm motility is dependent on a unique isoform of the Na,K-ATPase

The Na,K-ATPase, a member of the P-type ATPases, is composed of two subunits, alpha and beta, and is responsible for translocating Na(+) out of the cell and K(+) into the cell using the energy of hydrolysis of one molecule of ATP. The electrochemical gradient it generates is necessary for many cellular functions, including establishment of the plasma membrane potential and transport of sugars and ions in and out of the cell. Families of isoforms for both the alpha and beta subunits have been identified, and specific functional roles for individual isoforms are just beginning to emerge. The alpha4 isoform is the most recently identified Na, K-ATPase alpha isoform, and its expression has been found only in testis. Here we show that expression of the alpha4 isoform in testis is localized to spermatozoa and that inhibition of this isoform alone eliminates sperm motility. These data describe for the first time a biological function for the alpha4 isoform of the Na,K-ATPase, revealing a critical role for this isoform in sperm motility.     

Regulation of Na+, K+ -ATPase Isoforms in Rat Neostriatum by Dopamine and Protein Kinase C

Our previous studies showed that dopamine inhibits Na+,K+-ATPase activity in acutely dissociated neurons from striatum. In the present study, we have found that in this preparation, dopamine inhibited significantly (by approximately 25%) the activity of the alpha3 and/or alpha2 isoforms, but not the alpha1 isoform, of Na+,K+-ATPase. Dopamine, via D1 receptors, activates cyclic AMP-dependent protein kinase (PKA) in striatal neurons. Dopamine is also known to activate the calcium- and phospholipid-dependent protein kinase (PKC) in a number of different cell types. The PKC activator phorbol 12,13-dibutyrate reduced the activity of Na+,K+-ATPase alpha3 and/or alpha2 isoforms (by approximately 30%) as well as the alpha1 isoform (by approximately 15%). However, dopamine-mediated inhibition of Na+,K+-ATPase activity was unaffected by calphostin C, a PKC inhibitor. Dopamine did not affect the phosphorylation of Na+,K+-ATPase isoforms at the PKA-dependent phosphorylation site. Phorbol ester treatment did not alter the phosphorylation of alpha2 or alpha3 isoforms of Na+,K+-ATPase in neostriatal neurons but did increase the phosphorylation of the alpha1 isoform. Thus, in rat neostriatal neurons, treatment with either dopamine or PKC activators results in inhibition of the activity of specific (alpha3 and/or alpha2) isoforms of Na+,K+-ATPase, but this is not apparently mediated through direct phosphorylation of the enzyme. In addition, PKC is unlikely to mediate inhibition of rat Na+,K+-ATPase activity by dopamine in neostriatal neurons.     

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.     

Activity-Dependent Regulation of Na+,K+-ATPase α Isoform mRNA Expression In Vivo

To investigate the functional role of the different Na+, K(+)-ATPase alpha (catalytic) subunit isoforms in neuronal cells, we used quantitative in situ hybridization with riboprobes specific for alpha 1, alpha 2, and alpha 3 isoforms to measure the level of alpha isoform-specific expression in the neuroendocrine cells of the supraoptic (SON) and paraventricular (PVN) nuclei of rat hypothalamus. A prolonged increase in electrical activity of these cells, achieved by 5 days of salt treatment, increased the amount of alpha 1 isoform mRNA in the SON and PVN by 50%. Levels of alpha 1 mRNA in other brain regions and levels of alpha 2 and alpha 3 mRNAs were not affected by salt treatment. We conclude that the alpha 1 isoform Na+, K(+)-ATPase may be specifically adapted to pump out Na+, which enters the cells through voltage-gated channels during neuronal depolarization.     

Combined allosteric and competitive interaction between extracellular Na(+) and K(+) during ion transport by the alpha(1), alpha(2), and alpha(3) isoforms of the Na, K-ATPase

A combined allosteric and competitive model describes the interaction between extracellular Na(+) and Rb(+) during ion transport mediated by the Na, K-ATPase. The model was developed from experiments based on (86)Rb uptake by whole cells transfected with rat isoforms of the enzyme. In the absence of Na(+), only a single transport site for extracellular Rb(+) exists. After the occupation of the Na(+)-specific allosteric site, the Rb(+) transport pocket opens to allow occupation by an additional Rb(+) and the subsequent transport of the two Rb(+) ions into the cells. Na(+) can also directly compete with Rb(+) for binding to at least one of the transport sites. While the model derived here applies to each of the three rat isoforms of the Na, K-ATPase expressed in HeLa cells, subtle differences exist among the isoforms. The alpha(3)* isoform has an increased intrinsic affinity for Rb(+) and a lower affinity for the allosteric Na(+) site than alpha(1) or alpha(2)*. The stimulation of uptake observed according to the best-fit model is due to the displacement by Rb(+) of inhibitory Na(+) bound to the transport site.     

Expression of multiple Na+,K(+)-ATPase genes reveals a gradient of isoforms along the nonpigmented ciliary epithelium: functional implications in aqueous humor secretion.

The Na+,K(+)-ATPase alpha 1, alpha 2, and alpha 3 subunit isoforms have been shown to be differentially expressed in the nonpigmented (NPE) and pigmented (PE) cells of the ocular ciliary epithelium (CE) (Martin-Vasallo et al., J. Cell. Physiol., 141:243-252, 1989; Ghosh et al., J. Biol. Chem., 265:2935-2940, 1990). In this study we analyzed and compared the pattern of expression of the multiple Na+,K(+)-ATPase alpha (alpha 1, alpha 2, alpha 3) subunit genes with the pattern of expression of the Na+,K(+)-ATPase beta (beta 1, beta 2) subunit genes along the bovine CE. We have selected three regions in the CE, referred to as 1) the anterior region of the pars plicata, near the iris; 2) the middle region of the pars plicata; and 3) the posterior region of the pars plana, near the ora serrata. Using isoform-specific cDNA probes and antibodies for the Na+,K(+)-ATPase alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunits on Northern and Western blot analysis, we found that mRNA and polypeptides are expressed in all three CE regions with different abundance. The pattern of expression of alpha and beta isoforms detected along the NPE cell layers suggests a gradient of alpha 1, alpha 2, alpha 3, beta 1, and beta 2 mRNAs and polypeptides that correlates with decreasing Na+,K(+)-ATPase activity from the most anterior region at the pars plicata towards the posterior region at the ora serrata. We also found marked differences in the pattern of immunolocalization of Na+,K(+)-ATPase alpha 1, alpha 2, alpha 3, beta 1, and beta 2 subunit isoforms in different regions of the CE. In the anterior region, NPE cells stained intensely at the basal lateral membrane with specific monoclonal and polyclonal antibodies for each of the alpha (alpha 1, alpha 2, alpha 3) and beta (beta 1, beta 2) Na,K-ATPase isoforms. In the middle and posterior regions of the CE, NPE cells showed lower or absent levels of staining with alpha 1, alpha 2, alpha 3, and beta 1 antibodies, although staining with beta 2 was abundant. In contrast, PE cells throughout the CE were stained at the basal lateral membrane by antibodies to alpha 1 and beta 1, while no staining signals were detected with the rest of the antibodies (i.e. alpha 2, alpha 3, and beta 2). Our results support the conclusion that the three alpha and two beta isoforms of the Na+,K(+)-ATPase are differentially expressed in the two cell layers that make up the CE.(ABSTRACT TRUNCATED AT 250 WORDS)     

The human heart and rat brain IIA Na+ channels interact with different molecular regions of the beta1 subunit

The alpha subunit of voltage-gated Na(+) channels of brain, skeletal muscle, and cardiomyocytes is functionally modulated by the accessory beta(1), but not the beta(2) subunit. In the present study, we used beta(1)/beta(2) chimeras to identify molecular regions within the beta(1) subunit that are responsible for both the increase of the current density and the acceleration of recovery from inactivation of the human heart Na(+) channel (hH1). The channels were expressed in Xenopus oocytes. As a control, we coexpressed the beta(1)/beta(2) chimeras with rat brain IIA channels. In agreement with previous studies, the beta(1) extracellular domain sufficed to modulate IIA channel function. In contrast to this, the extracellular domain of the beta(1) subunit alone was ineffective to modulate hH1. Instead, the putative membrane anchor plus either the intracellular or the extracellular domain of the beta(1) subunit was required. An exchange of the beta(1) membrane anchor by the corresponding beta(2) subunit region almost completely abolished the effects of the beta(1) subunit on hH1, suggesting that the beta(1) membrane anchor plays a crucial role for the modulation of the cardiac Na(+) channel isoform. It is concluded that the beta(1) subunit modulates the cardiac and the neuronal channel isoforms by different molecular interactions: hH1 channels via the membrane anchor plus additional intracellular or extracellular regions, and IIA channels via the extracellular region only.     

Regions of the catalytic alpha subunit of Na,K-ATPase important for functional interactions with FXYD 2

The gamma modulator (FXYD 2) is a member of the FXYD family of single transmembrane proteins that modulate the kinetic behavior of Na,K-ATPase. This study concerns the identification of regions in the alpha subunit that are important for its functional interaction with gamma. An important effect of gamma is to increase K+ antagonism of cytoplasmic Na+ activation apparent as an increase in KNa' at high [K+]. We show that although gamma associates with alpha1, alpha2, and alpha3 isoforms, it increases the KNa' of alpha1 and alpha3 but not alpha2. Accordingly, chimeras of alpha1 and alpha2 were used to identify regions of alpha critical for the increased KNa'. As with alpha1 and alpha2, all chimeras associate with gamma. Kinetic analysis of alpha2front/alpha1back chimeras indicate that the C-terminal (Lys907-Tyr1018) region of alpha1, which includes transmembrane (TM)9 close to gamma, is important for the increase in KNa'. However, similar experiments with alpha1front/alpha2back chimeras indicate a modulatory role of the loop between TMs 7 and 8. Thus, as long as the alpha1 L7/8 loop is present, replacement of TM9 of alpha1 with that of alpha2 does not abrogate the gamma effect on KNa'. In contrast, as long as TM9 is that of alpha1, replacement of L7/8 of alpha1 with that of alpha2 does not abolish the effect. It is suggested that structural association of the TM regions of alpha and FXYD 2 is not the sole determinant of this effect of FXYD on KNa' but is subject to long range modulation by the extramembranous L7/8 loop of alpha.     

Genetic profiling reveals global changes in multiple biological pathways in the hearts of Na, K-ATPase alpha 1 isoform haploinsufficient mice.

The Na,K-ATPase transports three sodium ions out of the cell and two potassium ions into the cell using ATP hydrolysis for energy. The ion gradient formed by the Na,K-ATPase contributes to the resting membrane potential, maintains cellular excitability and is important for glucose and amino acid uptake in the cell. The alpha1 catalytic isoform is expressed in virtually all cell types. We have previously examined cardiac physiology of mice lacking one copy of the alpha1 isoform gene of the Na,K-ATPase. The observation of reduced cardiac contractility in the alpha1 heterozygous mice was unexpected since mice heterozygous for the alpha2 isoform displayed enhanced cardiac contractility similar to what would be observed with cardiac glycoside treatment. We further examined hearts from alpha1 heterozygous mice to identify genomic responses to reduced Na,K-ATPase capacity. Using microarray analyses, we identified groups of genes whose expressions were perturbed in the alpha1 heterozygous hearts compared to wild-type. Known functional relationships of these genes suggest that multiple biological pathways are altered by alpha1 hemizygosity including activation of the renin-angiotensin system, changes in genes of energy metabolism and transport and elevated brain natriuretic peptide. This suggests that Na,K-ATPase alpha1 isoform activity may be required in numerous cellular processes.     

Cellular distribution and differential gene expression of the three alpha subunit isoforms of the Na,K-ATPase in the ocular ciliary epithelium.

We have investigated the localization and pattern of expression of the three alpha subunit isoforms of Na,K-ATPase in the transporting ciliary epithelium of the bovine eye. Using specific cDNA probes and antisera to the alpha 1, alpha 2, and alpha 3 isoforms of Na,K-ATPase, we demonstrated that mRNAs and polypeptides for the three distinct forms of the Na,K-ATPase alpha subunit (alpha 1, alpha 2, and alpha 3) were expressed in the ciliary epithelium in vivo. Immunochemical localization of the three alpha isoforms of Na,K-ATPase in two ultrastructurally different regions of the ciliary epithelium (namely, the pars plicata and pars plana) revealed that the three alpha isoforms of Na,K-ATPase were distributed in a distinct fashion in the basolateral plasma membrane domains of nonpigmented (NPE) and pigmented (PE) cells. The NPE cells in the pars plicata showed an immunoreactive signal to all the three alpha isoforms; in the pars plana, they showed immunoreactive signals only for the alpha 1 and alpha 2 isoforms but not for alpha 3. The PE cells, in both the pars plana and pars plicata regions, showed an immunoreactive signal only for the alpha 1 isoform; immunoreactive signals were not detected for alpha 2 and alpha 3. To verify the differential immunostaining patterns of NPE and PE cells, specific antibodies for each of the three alpha subunit isoforms of Na,K-ATPase were applied to immunoblots containing microsomal fractions from flow cytometric-sorted cells (NPE and PE). Our results indicate that alpha 1, alpha 2, and alpha 3 polypeptides were present in microsomal fractions of NPE cells of the pars plicata and pars plana and that the alpha 1 polypeptide was the only polypeptide present in the PE cells from both regions of the ciliary epithelium. These results also revealed that the alpha 3 isoform epitope recognized by the monoclonal antibody McB-X3.1 in the pars plicata is not readily accessible in the pars plana. A cell line was established from the ciliary epithelium of a bovine eye by viral transformation with simian virus 40. In culture, this cell line expressed all three alpha isoforms at the mRNA and polypeptide levels, suggesting that the line may have derived from the NPE layer.