Expression of an ouabain-resistant Na,K-ATPase in CV-1 cells after transfection with a cDNA encoding the rat Na,K-ATPase alpha 1 subunit

We have used a gene transfer system to investigate the relationship between expression of the rat Na,K-ATPase alpha 1 subunit gene and ouabain-resistant Na,K-ATPase activity. A cDNA clone encoding the entire rat Na,K-ATPase alpha 1 subunit was inserted into the expression vector pSV2neo. This construct (pSV2 alpha 1) conferred resistance to 100 microM ouabain to ouabain-sensitive CV-1 cells. Hybridization analysis of transfected clones revealed the presence of both rat-specific and endogenous Na,K-ATPase alpha 1 subunit DNA and mRNA sequences. A single form of highly ouabain-sensitive 86Rb+ uptake was detected in CV-1 cells, whereas two distinct classes of ouabain-inhibitable uptake were observed in transfectants. One class exhibited the high ouabain sensitivity of the endogenous monkey Na,K-ATPase, while the second class showed the reduced ouabain sensitivity characteristic of the rodent renal Na,K-ATPase. Examination of the ouabain-sensitive, sodium-dependent ATPase activity of the transfectants also revealed a low affinity component of Na,K-ATPase activity characteristic of the rodent kidney enzyme. These results suggest that expression of the rat alpha 1 subunit gene is directly responsible for ouabain-resistant Na,K-ATPase activity in transfected CV-1 cells.     

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.     

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.     

Ouabain sensitivity of the alpha 3 isozyme of rat Na,K-ATPase

The Na,K-ATPase of rat brainstem axolemma membranes contains two isozymes of its catalytic subunit, alpha 2 and alpha 3. To isolate the alpha 3 isozyme functionally, purified axolemma Na,K-ATPase was treated with trypsin. Immunoblot analysis of trypsin-treated Na,K-ATPase using isozyme-specific antibodies showed that alpha 3 was significantly more resistant to digestion than alpha 2. The trypsin-resistant alpha 3 isozyme fraction, devoid of alpha 2, contained 50-60% of the ATPase activity, and was inhibited by ouabain half-maximally at 0.13 microM. This indicates that the alpha 3 Na,K-ATPase isozyme has a high sensitivity to cardiac glycosides.     

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.     

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.     

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.     

Expression of functional Na,K-ATPase isozymes in normal human cardiac biopsies.

In human heart failure, disturbances in Ca2+ homeostasis are well known but the fate of the Na,K-ATPase isoforms (alpha1beta1, alpha2beta1 and alpha3beta1), the receptors for cardiac glycosides, still remains under study. Microsomes have been purified from non-failing human hearts. As judged by the sensitivities of Na,K-ATPase activity to ouabain (IC50 values: 7.0 +/- 2.5 and 81 +/- 11 nM), 3H-ouabain-binding measurements at equilibrium with and without 10 mM K+ and by a biphasic ouabain dissociation process, at least two finctionally active Na,K-ATPase isozymes coexist in normal human hearts. These are demonstrated as a very high- and a high affinity ouabain-binding site. The KD values are 3.6 +/- 1.6 nM and 17 +/- 6 nM, respectively. The two dissociation rate constants are 42 x 10(4) min(-1) and 360 x 10(-4) min(-1). Addition of 10 mM K+ ions shifted the respective KD values for ouabain from 3.6 +/- 1.6 to 20 +/- 5 nM and from 17 +/- 6 nM to 125 +/- 25 nM, respectively. The isozymes involved are identified by comparing these three pharmacological parameters to those of each alpha/beta-isozyme separately expressed in Xenopus oocytes (9). In human heart, the very high affinity site for ouabain is the alpha1beta1 dimer and the high affinity site is alpha2beta1.     

Identification of a region within the Na,K-ATPase alpha subunit that contributes to differential ouabain sensitivity.

To analyze determinants within the Na,K-ATPase alpha subunit that contribute to differential ouabain sensitivity, we constructed and expressed a panel of chimeric cDNA molecules between ouabain-resistant and ouabain-sensitive alpha subunit cDNAs. When introduced into ouabain-sensitive monkey CV-1 cells, ouabain-resistant rat alpha 1 subunit cDNA and chimeras in which the 5' end of ouabain-sensitive human alpha 1 or rat alpha 2 subunit cDNA was replaced by the 5' end of rat alpha 1 subunit cDNA conferred resistance to 100 microM ouabain. Monkey cells transfected with the reciprocal chimeras were unable to survive selection in 1 microM ouabain. Rat alpha 2 subunit cDNA and a chimera in which the 5' end of rat alpha 1 subunit cDNA was replaced by the 5' end of rat alpha 2 subunit cDNA conferred resistance to 0.5 microM ouabain. These results suggest that determinants of ouabain resistance reside within the amino-terminal portions of the rat alpha 1 and alpha 2 subunits. Expression of chimeric alpha subunit cDNAs should prove useful for elucidating the structural basis of Na,K-ATPase function.     

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.     

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.     

Anomalous mobilities of Na,K-ATPase alpha subunit isoforms in SDS-PAGE: identification by N-terminal sequencing.

Three isoforms of the alpha subunit of Na,K-ATPase, alpha 1, alpha 2, and alpha 3 have been characterized at the DNA, mRNA and protein levels. In admixtures, isoforms migrate as doublets (i.e. alpha 1 and another band originally designated alpha +, comprising alpha 2 + alpha 3) when analyzed by SDS-PAGE. As deduced from cDNA sequences their masses range from 111.7 to 112.6 kDa. With conventional protein standards, however, SDS-PAGE yields nominal masses of 85-105 kDa. In this system, the presence of a doublet that reacted with a polyclonal anti-Na,K-ATPase antibody in the kidney was interpreted as indicating two molecular or conformational species of the kidney alpha sub-unit (Siegel, G.J. and Desmond, T.J. (1989) J. Biol. Chem. 264, 4751-4754). We report that Na,K-ATPase purified from dog, guinea pig and rat kidney medulla or from rat brain, can yield two distinct bands when analyzed by SDS-PAGE or STS-PAGE, migrating between 85 and 105 kDa. An additional band migrating at 117 and 120 kDa appears often in enzyme purified from rat and guinea pig kidney medulla. The apparent molecular weights and relative intensities of these bands vary with temperature and duration of incubation during sample preparation. N-terminal sequencing and monospecific antibody probes revealed that the two distinct bands obtained from the kidney enzyme consist only of the alpha 1 isoform. The band appearing at 117-120 kDa also contains only the alpha 1 N-terminal sequence. In contrast, as reported earlier (Sweadner, K.J. (1979) J. Biol. Chem. 254, 6060-6067), the doublet seen in brain preparations consists of alpha 1 and alpha 2 or (alpha 2 + alpha 3). We conclude that monospecific antibody probes or N-terminal sequencing must be used to identify Na,K-ATPase isoforms by SDS- or STS-PAGE. In addition, gel conditions that may affect the mobilities of the isoforms are discussed.     

The Na,K-ATPase alpha4 gene (Atp1a4) encodes a ouabain-resistant alpha subunit and is tightly linked to the alpha2 gene (Atp1a2) on mouse chromosome 1.

We have isolated and characterized cDNA clones encoding the murine homologue of a putative fourth Na,K-ATPase alpha subunit isoform (alpha4). The predicted polypeptide is 1032 amino acids in length and exhibits 75% amino acid sequence identity to the rat alpha1, alpha2, and alpha3 subunits. Within the first extracellular loop, the alpha4 subunit is highly divergent from other Na,K-ATPase alpha subunits. Because this region of Na,K-ATPase is a major determinant of ouabain sensitivity, we tested the ability of the rodent alpha4 subunit to transfer ouabain resistance in a transfection protocol. We find that a cDNA containing the complete rodent alpha4 ORF is capable of conferring low levels of ouabain resistance upon HEK 293 cells, an indication that the alpha4 subunit can substitute for the endogenous ouabain-sensitive alpha subunit of human cells. Nucleotide sequences specific for the murine alpha4 subunit were used to identify the chromosomal position of the alpha4 subunit gene. By hybridizing an alpha4 probe with a series of BACs, we localized the alpha4 subunit gene (Atp1a4) to the distal portion of mouse chromosome 1, in very close proximity to the murine Na,K-ATPase alpha2 subunit gene. In adult mouse tissues, we detected expression of the alpha4 subunit gene almost exclusively in testis, with low levels of expression in epididymis. The close similarities in the organization and expression pattern of the murine and human alpha4 subunit genes suggest that these two genes are orthologous. Together, our studies indicate that the alpha4 subunit represents a functional Na,K-ATPase alpha subunit isoform.     

Modulation of the Na,K-pump function by beta subunit isoforms

To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K- pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+.     

Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector.

Site-directed mutagenesis was used to identify residues responsible for the greater than 1,000-fold difference in ouabain sensitivity between the rat Na,K-ATPase alpha 1 and alpha 2 isoforms. A series of mutagenized cDNAs was constructed that replaced residues of the rat alpha 2 subunit with the corresponding residues from the rat alpha 1 subunit. These cDNAs were cloned into a mammalian episomal expression vector (EBOpLPP) and expressed in ouabain-sensitive primate cells. Either of two single substitutions introduced into the rat alpha 2 subunit cDNA (Leu-111----Arg or Asn-122----Asp) conferred partial resistance (approximately 10 microM ouabain) upon transformed cells. This resistance was intermediate between the levels conferred by the rat alpha 1 cDNA (approximately 500 microM ouabain) and the rat alpha 2 cDNA (approximately 0.2 microM ouabain). A double substitution of the rat alpha 2 cDNA (Leu-111----Arg and Asn-122----Asp) conferred a resistance level equivalent to that obtained with rat alpha 1. These results demonstrate that the residues responsible for isoform-specific differences in ouabain sensitivity are located at the end of the H1-H2 extracellular domain. The combination of site-directed mutagenesis and episomal expression provides a useful system for the selection and analysis of mutants.     

The alpha4 isoform of the Na,K-ATPase is expressed in the germ cells of the testes.

In addition to the three isoforms of the catalytic subunit of the Na, K-ATPase originally identified (alpha1, alpha2, and alpha3), a fourth alpha polypeptide (alpha4) has recently been found in mammalian cells. This novel alpha-subunit of the Na,K-ATPase is selectively expressed in male gonadal tissues. In the testes, alpha4 is functionally active and comprises approximately half of the Na, K-ATPase activity of the organ. At present, the pattern of expression of the alpha4 polypeptide within the cells of the male gonad is unknown. By in situ hybridization, immunocytochemistry, and the ouabain inhibition profile of Na,K-ATPase activity, we show that the alpha4-subunit is expressed in the germ cells of rat testes. The highest amounts of the isoform are found in spermatozoa, where it constitutes two thirds of the Na,K-ATPase activity of the gametes. The other Na pump present in the cells is the ubiquitously expressed alpha1 polypeptide. The characteristic localization of alpha4 in the gonad is further supported by the drastic reduction of the polypeptide in mice that are infertile as a consequence of arrest in maturation of the germ cells. In addition, GC-1spg cells, a murine cell line derived from testis spermatogonia, also contain the Na, K-ATPase alpha4 polypeptide. However, the level of expression of the isoform in these cells is much lower than in the spermatozoa, a fact that may depend on the limited ability of the GC-1spg cells to differentiate in vitro. The particular expression of the Na,K-ATPase alpha4 isoform we encounter and the specific enzymatic properties of the polypeptide suggests its importance for ionic homeostasis of the germ cells of the testes.     

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.     

Identification of a specific role for the Na,K-ATPase alpha 2 isoform as a regulator of calcium in the heart.

It is well accepted that inhibition of the Na,K-ATPase in the heart, through effects on the Na/Ca exchanger, raises the intracellular Ca2+ concentration and strengthens cardiac contraction. However, the contribution that individual isoforms make to this calcium regulatory role is unknown. Assessing the phenotypes of mouse hearts with genetically reduced levels of Na,K-ATPase alpha 1 or alpha 2 isoforms clearly demonstrates different functional roles for these isoforms in vivo. Heterozygous alpha 2 hearts are hypercontractile as a result of increased calcium transients during the contractile cycle. In contrast, heterozygous alpha 1 hearts are hypocontractile. The different functional roles of these two isoforms are further demonstrated since inhibition of the alpha 2 isoform with ouabain increases the contractility of heterozygous alpha 1 hearts. These results definitively illustrate a specific role for the alpha 2 Na,K-ATPase isoform in Ca2+ signaling during cardiac contraction.     

Postnatal changes in Na,K-ATPase isoform expression in rat cardiac ventricle. Conservation of biphasic ouabain affinity.

The cardiac glycoside sensitivity of the rat heart changes during postnatal maturation and in response to certain pathological conditions. The Na,K-ATPase is thought to be the receptor for cardiac glycosides, and there are three isozymes of its catalytic (alpha) subunit with different cardiac glycoside affinities: alpha 1 (low affinity) and alpha 2 and alpha 3 (high affinity). We examined the developmental expression of the alpha subunit isozymes in rat ventricular membrane preparations by immunoblotting with isozyme-specific antibodies. The alpha 1 isozyme was present throughout all stages of maturation. A developmental switch from alpha 3 to alpha 2 occurred between 14 and 21 days after birth. Measurements of [3H]ouabain binding and inhibition of Na,K-ATPase activity indicated that alpha 2 and alpha 3 should make equivalent contributions to ion pump capacity; in both neonatal natal and adult preparations, ouabain interacted with a single class of high-affinity binding sites (KD = 15 or 40 nM, respectively; Bmax = 4-5 pmol/mg protein), and at low concentrations produced a similar degree of Na,K-ATPase inhibition (25%). The results indicate that the developmental difference in cardiac glycoside sensitivity cannot be explained by quantitative differences in the proportion of high-affinity isozymes of the Na,K-ATPase. The switch from alpha 3 to alpha 2 coincides with other major changes in cardiac electrophysiology and calcium metabolism.