Expression of Na,K-ATPase alpha subunit isoforms in the human ciliary body and cultured ciliary epithelial cells

We have analyzed the expression of Na,K-ATPase alpha subunit isoforms in the transporting ciliary processes of the human eye and in cultured cells derived from non-pigmented (NPE) and pigmented (PE) ciliary epithelium. Northern hybridization analysis shows that the mRNAs encoding all the three distinct forms of Na,K-ATPase alpha subunit [alpha 1, alpha 2, and alpha 3] are expressed in the human ciliary processes in vivo. Immunohistochemical analysis using antibodies specific for each of the three alpha subunit isoforms confirms that these polypeptides are present in the microsomal fraction from the human ciliary processes. The monoclonal antibody McB2, which is specific to the Na,K-ATPase alpha 2 subunit isoform, has been found to decorate specifically the basolateral membrane domains of NPE cells but not of the PE cells, suggesting its expression in vivo only in the ocular NPE ciliary epithelium. However, cultured cells derived from the NPE and PE layers exhibit a different pattern of expression of mRNA and protein for the Na,K-ATPase alpha subunit isoforms when compared to the tissue. Both the NPE and PE cells express alpha 1 and alpha 3 mRNA and polypeptide, whereas alpha 2 mRNA and polypeptide are undetectable in these cells. The established cell lines derived from the NPE layer express comparable levels of the alpha 1 and alpha 3 isoforms of Na,K-ATPase as detected in the primary culture. However, the established NPE cell lines are also distinguishable from the normal PE cells when analyzed by Western blot analysis with A x 2 antibodies. The results presented here clearly show that the NPE and PE cells in the ciliary body have a distinct expression of Na,K-ATPase alpha subunit isoforms as compared to cultured cells.     

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.     

RT-PCR detection of the Na,K-ATPase beta3-isoform in human heart.

The Na,K-ATPase is a heterodimer composed of an alpha-catalytic and a beta-glycoprotein subunit. At present, three different alpha-polypeptides (alpha1, alpha2, alpha3) and two distinct beta-isoforms (beta1 and beta2) have been detected in human heart. The aim of the present study was to determine whether or not the beta3-isoform of the Na,K-ATPase can be detected in human heart. Using the highly sensitive method of RT-PCR, we here show that human heart expresses the beta3-isoform of the Na,K-ATPase. Given the differences in pharmacological properties of the nine different Na,K-ATPase isoenzymes (containing all combinations of the subunit isoforms), the study of beta3-isoform regulation in human heart may be of interest in understanding the altered response of human myocardium to digitalis therapy during heart failure.     

Subcellular distribution and immunocytochemical localization of Na,K-ATPase subunit isoforms in human skeletal muscle

Summary

The expression of Na,K-ATPase isoforms was investigated in human skeletal muscle membranes isolated by subcellular fractionation. The α1, α2, α3 and β1 subunits were detectable in membranes prepared from the human soleus muscle. The α1 subunit was largely detected in a fraction enriched with plasma membranes (PM), its abundance in an Intracellular membrane fraction (IM) accounted for only 4% of that in the PM fraction. No α1 subunits were detected in membranes of sarcoplasmic reticulum (SR) origin. The PM and IM fractions were enriched with α2 subunits which were less abundant in the SR-enriched fraction. The abundance of α2 molecules within the IM fraction was about 75% of that in the PM fraction when the total protein content for the two fractions was taken into account. Immuno-cytochemical studies confirmed the localization of the α1 subunit to the muscle cell surface. The α2 subunit was also found to be present in the cell surface but the observation that α2 immuno-fluorescence was diffusely dispersed throughout the muscle fibre indicated that it was also present intracellularly, consistent with its biochemical localization in the PM and IM membrane fractions. The α3 subunit was detected largely in the PM fraction but the lack of good antibodies to this isoform precluded an analysis of its immunocytochemical localization. The β1 subunit was enriched in the PM fraction but was also detected to a modest extent in the IM. A polyclonal anti-β2 antibody, which reacted positively with both human brain microsomes and rat skeletal muscle membranes, revealed that human skeletal muscles contained no immunoreactive β2 subunits. Our results indicate that the human soleus expresses the α1 and α2 (and possibly the α3) subunits of the Na,K-ATPase and that the activity of these isoforms must be supported by the β1 subunit in this muscle.     

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.     

Human myocardial Na,K-ATPase concentration in heart failure

The Na,K-ATPase is of major importance for active ion transport across the sarcolemma and thus for electrical as well as contractile function of the myocardium. Furthermore, it is receptor for digitalis glycosides. In human studies of the regulatory aspects of myocardial Na,K-ATPase concentration a major problem has been to obtain tissue samples. Methodological accomplishments in quantification of myocardial Na,K-ATPase using vanadate facilitated ³H-ouabain binding to intact samples have, however, made it possible to obtain reliable measurements on human myocardial necropsies obtained at autopsy as well as on biopsies of a wet weight of only 1–2 mg obtained during heart catheterisation. However, access to the ultimately, normal, vital myocardial tissue has come from the heart transplantation programs, through which myocardial samples from cardiovascular healthy organ donors have become available. In the present paper we evaluate the various values reported for normal human myocardial Na,K-ATPase concentration, its regulation in heart disease and the association with digitalization. Normal myocardial Na,K-ATPase concentration level is found to be 700 pmol/g wet weight. No major variations were found between or within the walls of the heart ventricles. During the first few years of life a marked decrease in myocardial Na,K-ATPase concentration is followed by a stable level obtained in early adulthood and normally maintained throughout life. In patients with enlarged cardiac x-ray silhouette a significant positive, linear correlation between left ventricular ejection fraction (EF) and Na,K-ATPase concentration was established. A maximum reduction in Na,K-ATPase concentration of 89% was obtained when EF was reduced to 20%. Generally, heart failure associated with heart dilatation, myocardial hypertrophy as well as ischaemic heart disease is associated with reductions in myocardial Na,K-ATPase concentration of around 25%. During digoxin treatment of heart failure patients a further reduction in functional myocardial Na,K-ATPase concentration of 15% has been found. Thus, the total reduction in functional myocardial Na,K-ATPase concentration in digitalised heart failure patients may well be of the magnitude 40%. In conclusion, it has become possible to quantify human myocardial Na,K-ATPase in health and disease. Revealed reductions are in heart failure of importance for contractile function, generation of arrhythmia and for digoxin treatment.     

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.     

Expression of the Avian Na,K-ATPase Subunits in Dictyostelium discoideum

This study explored whether Dictyostelium discoideum can be used to express the avian Na,K-ATPase, a heterodimeric membrane protein. Dictyostelium was able to express mRNAs encoding the avian Na,K-ATPase subunits. However, Dictyostelium expressed avian Na,K-ATPase protein when only when a Dictyostelium consensus ribosomal binding sequence, AAAATAAA, was inserted in front of the open reading frames of the α₁- and β₁-subunit cDNAs and the first eight codons following the start-translation codons were changed to Dictyostelium preferred codons. These modified mRNAs appeared to be much less stable than the forms that were not readily translated. Dictyostelium could express the avian β-subunit alone but only expressed the α₁-subunit when the β₁-subunit was co-expressed. Subunit assembly occurred in cells expressing both α₁- and β₁-subunits. The bulk of the exogenously expressed sodium pump subunits remained in an intracellular compartment, presumed to be the endoplasmic reticulum. Dictyostelium exported little or no Na,K-ATPase or free β-subunit to the plasma membrane. Received: 7 July 1998/Revised: 8 October 1998     

Molecular cloning and sequence analysis of human Na,K-ATPase beta-subunit.

We have isolated a cDNA clone for the beta-subunit of HeLa cell Na,K-ATPase, containing a 2208-base-pair cDNA insert covering the whole coding region of the beta-subunit. Nucleotide sequence analysis revealed that the amino acid sequence of human Na,K-ATPase exhibited 61% homology with that of Torpedo counterpart (Noguchi et al. (1986) FEBS Lett. in press). A remarkable conservation in the nucleotide sequence of the 3' non-coding region was detected between the human and Torpedo cDNAs. RNA blot hybridization analysis revealed the presence of two mRNA species in HeLa cells. S1 nuclease mapping indicated that they were derived from utilization of two distinct polyadenylation signals in vivo. Total genomic Southern hybridization indicated the existence of only a few, possibly one set of gene encoding the Na,K-ATPase beta-subunit in the human genome.     

The Na,K-ATPase

The energy dependent exchange of cytoplasmic Na⁺ for extracellular K⁺ in mammalian cells is due to a membrane bound enzyme system, the Na,K-ATPase. The exchange sustains a gradient for Na⁺ into and for K⁺ out of the cell, and this is used as an energy source for creation of the membrane potential, for its de- and repolarisation, for regulation of cytoplasmic ionic composition and for transepithelial transport. The Na,K-ATPase consists of two membrane spanning polypeptides, an -subunit of 112-kD and a -subunit, which is a glycoprotein of 35-kD. The catalytic properties are associated with the -subunit, which has the binding domain for ATP and the cations. In the review, attention will be given to the biochemical characterization of the reaction mechanism underlying the coupling between hydrolysis of the substate ATP and transport of Na⁺ and K⁺.     

Cation selectivity of gastric H,K-ATPase and Na,K-ATPase chimeras.

Chimeras of the catalytic subunits of the gastric H,K-ATPase and Na, K-ATPase were constructed and expressed in LLC-PK1 cells. The chimeras included the following: (i) a control, H85N (the first 85 residues comprising the cytoplasmic N terminus of Na,K-ATPase replaced by the analogous region of H,K-ATPase); (ii) H85N/H356-519N (the N-terminal half of the cytoplasmic M4-M5 loop also replaced); and (iii) H519N (the entire front half replaced). The latter two replacements confer a decrease in apparent affinity for extracellular K+. The 356-519 domain and, to a greater extent, the H519N replacement confer increased apparent selectivity for protons relative to Na+ at cytoplasmic sites as shown by the persistence of K+ influx when the proton concentration is increased and the Na+ concentration decreased. The pH and K+ dependence of ouabain-inhibitable ATPase of membranes derived from the transfected cells indicate that the H519N and, to a lesser extent, the H356-519N substitution decrease the effectiveness of K+ to compete for protons at putative cytoplasmic H+ activation sites. Notable pH-independent behavior of H85N/H356-519N at low Na+ suggests that as pH is decreased, Na+/K+ exchange is replaced largely by (Na+ + H+)/K+ exchange. With H519N, the pH and Na+ dependence of pump and ATPase activities suggest relatively active H+/K+ exchange even at neutral pH. Overall, this study provides evidence for important roles in cation selectivity for both the N-terminal half of the M4-M5 loop and the adjacent transmembrane helice(s).     

Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications

We have characterized cDNAs coding for three Na,K-ATPase alpha subunit isoforms from the rat, a species resistant to ouabain. Northern blot and S1-nuclease mapping analyses revealed that these alpha subunit mRNAs are expressed in a tissue-specific and developmentally regulated fashion. The mRNA for the alpha 1 isoform, approximately equal to 4.5 kb long, is expressed in all fetal and adult rat tissues examined. The alpha 2 mRNA, also approximately equal to 4.5 kb long, is expressed predominantly in brain and fetal heart. The alpha 3 cDNA detected two mRNA species: a approximately equal to 4.5 kb mRNA present in most tissues and a approximately equal to 6 kb mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the alpha subunits are encoded by a multigene family. Structural analysis of the alpha subunits from rat and other species predicts a polytopic protein with seven membrane-spanning regions. Isoform diversity of the alpha subunit may provide a biochemical basis for Na,K-ATPase functional diversity.     

Na,K-ATPase is essential for embryonic heart development in the zebrafish

Na,K-ATPase is an essential gene maintaining electrochemical gradients across the plasma membrane. Although previous studies have intensively focused on the role of Na,K-ATPase in regulating cardiac function in the adults, little is known about the requirement for Na,K-ATPase during embryonic heart development. Here, we report the identification of a zebrafish mutant, heart and mind, which exhibits multiple cardiac defects, including the primitive heart tube extension abnormality, aberrant cardiomyocyte differentiation, and reduced heart rate and contractility. Molecular cloning reveals that the heart and mind lesion resides in the alpha1B1 isoform of Na,K-ATPase. Blocking Na,K-ATPase alpha1B1 activity by pharmacological means or by morpholino antisense oligonucleotides phenocopies the patterning and functional defects of heart and mind mutant hearts, suggesting crucial roles for Na,K-ATPase alpha1B1 in embryonic zebrafish hearts. In addition to alpha1B1, the Na,K-ATPase alpha2 isoform is required for embryonic cardiac patterning. Although the alpha1B1 and alpha2 isoforms share high degrees of similarities in their coding sequences, they have distinct roles in patterning zebrafish hearts. The phenotypes of heart and mind mutants can be rescued by supplementing alpha1B1, but not alpha2, mRNA to the mutant embryos, demonstrating that alpha1B1 and alpha2 are not functionally equivalent. Furthermore, instead of interfering with primitive heart tube formation or cardiac chamber differentiation, blocking the translation of Na,K-ATPase alpha2 isoform leads to cardiac laterality defects.     

Effects of tetradecyl sulfate on electrophoretic resolution of kidney Na,K-ATPase catalytic subunit isoforms

Inclusion of sodium tetradecyl sulfate in the Laemmli polyacrylamide gel electrophoresis system produces resolution of the kidney Na,K-ATPase catalytic subunit into a doublet and sharpens demarcation of catalytic subunit isoforms in NA,K-ATPase from brain.     

Na,K-ATPase function in alternating electric fields.

Alternating currents affect ion transport processes and ATP splitting through changes in the activation of the membrane Na,K-ATPase. Both processes vary with the frequency, and the effective range includes the environmental 60 Hz. ATP splitting by Na,K-ATPase suspensions decreases for the enzyme under normal conditions, with the maximum effect at 100 Hz. ATP splitting increases when the enzyme activity is lowered to less than half its optimal value by changes in temperature, ouabain concentration, etc. These observations can be explained by the effects of the ionic currents on ion binding at the enzyme activation sites. Such a mechanism could account for the effects of electromagnetic fields on cells, as the transmembrane enzyme can convey the effect of an extracellular signal into the cell via ionic fluxes, and the measured threshold field is within the range of reported biological effects.     

Cysteine protects Na,K-ATPase and isolated human lymphocytes from silver toxicity.

Metal-binding proteins are important components of retroviruses such as human immunodeficiency virus (HIV). Therefore, metals could be used as antiviral agents. However, most metals are toxic for humans with the exception of silver which is toxic only to prokaryotic cells and viruses. In addition, HIV infection causes a decrease in body cysteine. We formed a complex of silver and cysteine, named silver-cysteine. Healthy human lymphocytes were incubated with silver-nitrate or silver-cysteine. Negligible cell survival was seen at 50 microM silver-nitrate. However, in presence of 1 mM cysteine, the viability remained unaffected up to 1 mM of silver. Further, silver inhibition of isolated Na,K-ATPase was easily reversed by cysteine. Thus, non-toxic silver-cysteine could be used as an anti-viral and cysteine-replenishing agent.     

Calmodulin increases Ca-dependent inhibition of the Na,K-ATPase in human red blood cells.

Proteins in human red cell hemolysate were purified to determine which of them increase inhibition of the Na,K-ATPase in the presence of 2 microM free Ca. Samples purified 600,000-fold inhibited the Na,K-ATPase of human red cells in a Ca-dependent manner and stimulated the (Ca+Mg)-ATPase. These samples contained two proteins as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): calmodulin (18,000 Mr), which comprised most (greater than 90%) of the total protein, and an unidentified protein of approximately 13,000 Mr. Both proteins were a distinctive light yellow when stained with silver. Calmodulin from bovine testes also inhibited the Na,K-ATPase and stimulated the (Ca+Mg)-ATPase. This preparation also contained two proteins as analyzed by SDS-PAGE: calmodulin (95 to 99% of the total protein) and another protein of approximately 13,000 Mr (1 to 5% of the total protein). Both were light yellow when stained with silver. Since the amount of red cell protein was limited, the remainder of the study was carried out with the bovine testes preparation. Heating the testes preparation decreased, but did not abolish, inhibition of the Na,K-ATPase and reduced stimulation of the (Ca+Mg)-ATPase. When corrected for denatured calmodulin, both heated and unheated proteins increased inhibition of the Na,K-ATPase to the same extent. The Na,K-ATPase was inhibited at 2 microM free Ca in a dose-dependent manner over a range of 15 to 100 nM calmodulin. To establish if the inhibition was due to the calmodulin or the 13,000 Mr protein, both were electroeluted after SDS-PAGE. Electroeluted calmodulin stimulated the (Ca+Mg)-ATPase and increased Ca inhibition of the Na,K-ATPase. Electroeluted amounts of the smaller Mr protein slightly stimulated the (Ca+Mg)-ATPase, but had no effect on the Na,K-ATPase. This protein was digested with cyanogen bromide, partially sequenced, and thereby identified as a fragment of calmodulin. We conclude that intact calmodulin increases inhibition of the Na,K-ATPase at 2 microM free Ca. We suggest that calmodulin is part of a mechanism mediating the effects of physiological free Ca on the Na,K-ATPase.     

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.