Immunochemical demonstration of a novel beta-subunit isoform of X, K-ATPase in human skeletal muscle

Recently we have identified mRNA encoding a hitherto unknown mammalian X,K-ATPase beta-subunit expressed predominantly in muscle tissue (Pestov, N. B. et al. (1999) FEBS Lett. 456, 243-248). Here we demonstrate the existence of the predicted protein, designated as beta(m) (beta(muscle)), in human adult skeletal muscle membranes using immunoblotting with beta(m)-specific antibodies generated against recombinant polypeptide formed by extramembrane beta(m) domains. The electrophoretic mobility of beta(m) was shown to be abnormally low due to the presence of Glu-rich sequences. In contrast to mature forms of other known X,K-ATPase beta-subunits, carbohydrate moiety of beta(m) is sensitive to endoglycosidase H and appears to be composed of short high-mannose or hybrid N-glycans. This finding argues in favor of an intracellular location of beta(m) in human skeletal muscle.     

Identification of a novel gene of the X,K-ATPase beta-subunit family that is predominantly expressed in skeletal and heart muscles.

We have identified the fifth member of the mammalian X,K-ATPase beta-subunit gene family. The human and rat genes are largely expressed in skeletal muscle and at a lower level in heart. The deduced human and rat proteins designated as beta(muscle) (beta(m)) consist of 357 and 356 amino acid residues, respectively, and exhibit 89% identity. The sequence homology of beta(m) proteins with known Na,K- and H,K-ATPase beta-subunits are 30.5-39.4%. Unlike other beta-subunits, putative beta(m) proteins have large N-terminal cytoplasmic domains containing long Glu-rich sequences. The data obtained indicate the existence of hitherto unknown X,K-ATPase (most probably Na,K-ATPase) isozymes in muscle cells.     

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.     

Examination of the calcium-modulated protein S100 alpha and its target proteins in adult and developing skeletal muscle.

In this study radioimmunoassay, immunohistochemistry, Northern blot analysis, and a gel overlay technique have been used to examine the level, subcellular distribution, and potential target proteins of the S100 family of calcium-modulated proteins in adult and developing rat skeletal muscles. Adult rat muscles contained high levels of S100 proteins but the particular form present was dependent on the muscle type: cardiac muscle contained exclusively S100 alpha, slow-twitch skeletal muscle fibers contained predominantly S100 alpha, vascular smooth muscle contained both S100 alpha and S100 beta, and fast-twitch skeletal muscle fibers contained low but detectable levels of S100 alpha and S100 beta. While the distribution of S100 mRNAs paralled the protein distribution in all muscles there was no direct correlation between the mRNA and protein levels in different muscle types, suggesting that S100 protein expression is differentially regulated in different muscle types. Immunohistochemical analysis of the cellular distribution of S100 proteins in adult skeletal muscles revealed that S100 alpha staining was associated with muscle cells, while S100 beta staining was associated with nonmuscle cells. Radioimmunoassays of developing rat skeletal muscles demonstrated that all developing muscles contained low levels of S100 alpha at postnatal day 1 and that as development proceeded the S100 alpha levels increased. In contrast to adult muscle S100 alpha expression was confined to fast-twitch fibers in developing skeletal muscle until postnatal day 21. At postnatal day 1, developing contractile elements were S100 alpha positive, but no staining periodicity was detectable. At postnatal day 21, S100 alpha exhibited the same subcellular localization as seen in the adult: colocalization with the A-band and/or longitudinal sarcoplasmic reticulum. Comparison of the S100 alpha-binding protein profiles in fast- and slow-twitch fibers of various species revealed few, if any, species- or fiber type-specific S100 binding proteins. Isolated sarcoplasmic reticulum fractions and myofibrils contained multiple S100 alpha-binding proteins. The colocalization of S100 alpha and S100 alpha-binding proteins with the contractile apparatus and sarcoplasmic reticulum suggest that S100 alpha may regulate excitation and/or contraction in slow-twitch fibers.     

Tyrosine-phosphorylated and nonphosphorylated sodium channel beta1 subunits are differentially localized in cardiac myocytes

Voltage-gated sodium channel alpha and beta subunits expressed in mammalian heart are differentially localized to t-tubules and intercalated disks. Sodium channel beta subunits are multifunctional molecules that participate in channel modulation and cell adhesion. Reversible, receptor-mediated changes in beta1 tyrosine phosphorylation modulate its ability to recruit and associate with ankyrin. The purpose of the present study was to test our hypothesis that tyrosine-phosphorylated beta1 (pYbeta1) and nonphosphorylated beta1 subunits may be differentially localized in heart and thus interact with different cytoskeletal and signaling proteins. We developed an antibody that specifically recognizes pYbeta1 and investigated the differential subcellular localization of beta1 and pYbeta1 in mouse ventricular myocytes. We found that pYbeta1 colocalized with connexin-43, N-cadherin, and Nav1.5 at intercalated disks but was not detected at the t-tubules. Anti-pYbeta1 immunoprecipitates N-cadherin from heart membranes and from cells transfected with beta1 and N-cadherin in the absence of other sodium channel subunits. pYbeta1 does not associate with ankyrinB in heart membranes. N-cadherin and connexin-43 associate with Nav1.5 in heart membranes as assessed by co-immunoprecipitation assays. We propose that sodium channel complexes at intercalated disks of ventricular myocytes are composed of Nav1.5 and pYbeta1 and that these complexes are in close association with both N-cadherin and connexin-43. beta1 phosphorylation appears to regulate its localization to differential subcellular domains.     

RT-PCR detection of Na,K-ATPase subunit isoforms in human umbilical vein endothelial cells (HUVEC): evidence for the presence of alpha1 and beta3.

The endothelial Na,K-ATPase is an active component in maintaining a variety of normal vascular functions. The enzyme is characterized by a complex molecular heterogeneity that results from differential expression and association of multiple isoforms of both its alpha- and beta-subunits. The aim of the present study was to determine which isoforms of the Na,K-ATPase are expressed in human endothelial cells. HUVEC (human umbilical vein endothelial cells) were used as a model of well known human endothelial cells. The high sensitive method RT-PCR was used with primers specific for the various isoforms of the alpha- and beta-subunits of the Na,K-ATPase. The results show that HUVEC express alpha1-, but not alpha2-, alpha3- or alpha4-isoforms of the catalytic subunit and that beta3- but not beta2- or beta1-isoforms is present in these cells. These findings are in contradiction with our previous detection of Na,K-ATPase isoforms in HUVEC using antibodies (14). Such results raise the technical problem of the specificity of the available antibodies directed against the different isoforms as well as the question of the physiological relevance of the diversity of the Na,K-ATPase isoforms.     

Beta 1- and beta 3-subunits can associate with presynthesized alpha-subunits of Xenopus oocyte Na,K-ATPase.

Oligomerization of newly synthesized alpha- and beta-subunits is a prerequisite for the structural and functional maturation of Na,K-ATPase. In this study, we have tested the competence of presynthesized alpha- and beta-subunits to assemble into functional enzyme complexes. Antisense oligonucleotides complementary to alpha-mRNA were used to inhibit alpha-subunit synthesis in Xenopus oocytes leaving a presynthesized trypsin-sensitive alpha-subunit pool. beta-Subunits expressed in these oocytes from injected cRNA assembled with the preexisting alpha-subunits, rendered them trypsin-resistant, and permitted the expression of more ouabain binding sites at the plasma membrane. Similarly, presynthesized beta 1- or beta 3-subunits produced in Xenopus oocytes by injection of beta-cRNA and later of specific antisense oligonucleotides were stabilized and transported out of the endoplasmic reticulum when alpha-cRNA was injected into oocytes. These data indicate that alpha- and beta-subunits can insert into endoplasmic reticulum membranes independent of each other in an assembly-competent form and retain their ability for oligomerization after synthesis.     

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

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

Betam,a structural member of the X,K-ATPase beta subunit family,resides in the ER and does not associate with any known X,K-ATPase alpha subunit

betam, a muscle-specific protein, is structurally closely related to the X,K-ATPase beta subunits, but its intrinsic function is not known. In this study, we have expressed betam in Xenopus oocytes and have investigated its biosynthesis and processing as well as its putative role as a chaperone of X,K-ATPase alpha subunits, as a regulator of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), or as a Ca(2+)-sensing protein. Our results show that betam is stably expressed in the endoplasmic reticulum (ER) in its core glycosylated, partially trimmed form. Both full-length betam, initiated at Met(1), and short betam species, initiated at Met(89), are detected in in vitro translations as well as in Xenopus oocytes. betam cannot associate with and stabilize Na,K-ATPase (NK), or gastric and nongastric H,K-ATPase (HK) alpha isoforms. betam neither assembles stably with SERCA nor is its trypsin sensitivity or electrophoretic mobility influenced by Ca(2+). A mutant, in which the distinctive Glu-rich regions in the betam N-terminus are deleted, remains stably expressed in the ER and can associate with, but not stabilize X,K-ATPase alpha subunits. On the other hand, a chimera in which the ectodomain of betam is replaced with that of beta1 NK associates efficiently with alpha NK isoforms and produces functional Na,K-pumps at the plasma membrane. In conclusion, our results indicate that betam exhibits a cellular location and functional role clearly distinct from the typical X,K-ATPase beta subunits.     

The hormonal regulation of mRNA Na,K-ATPase expression in rat kidneys in postnatal ontogeny]

A study was made of effects of aldosterone, aldosterone+dexamethasone, and aldosterone+spironolactone on Na,K-ATPase mRNA expression in renal cortex of adult and 10 day old rats, when kidney is not sensitive to the hormone injection. It is shown that hormonal induction of synthesis of Na,K-pump mRNA occurs in the early postnatal period apart from mineral corticoid receptors. It seems probable that aldosterone exerts its action in 10 day old rats by interaction with glucocorticoid receptors inducing synthesis of different amounts of alpha- and beta-subunits of Na,K-ATPase.     

An ion-transporting ATPase encodes multiple apical localization signals

Epithelial cells accumulate distinct populations of membrane proteins at their two plasmalemmal domains. We have examined the molecular signals which specify the differential subcellular distributions of two closely related ion pumps. The Na,K-ATPase is normally restricted to the basolateral membranes of numerous epithelial cell types, whereas the H,K-ATPase is a component of the apical surfaces of the parietal cells of the gastric epithelium. We have expressed full length and chimeric H,K-ATPase/Na,K-ATPase cDNAs in polarized renal proximal tubular epithelial cells (LLC-PK1). We find that both the alpha and beta subunits of the H,K-ATPase encode independent signals that specify apical localization. Furthermore, the H,K-ATPase beta-subunit possesses a sequence which mediates its participation in the endocytic pathway. The interrelationship between epithelial sorting and endocytosis signals suggested by these studies supports the redefinition of apical and basolateral as functional, rather than simply topographic domains.     

A pump-independent function of the Na,K-ATPase is required for epithelial junction function and tracheal tube-size control

The heterodimeric Na,K-ATPase has been implicated in vertebrate and invertebrate epithelial cell junctions, morphogenesis and oncogenesis, but the mechanisms involved are unclear. We previously showed that the Drosophila Na,K-ATPase is required for septate junction (SJ) formation and that of the three beta-subunit loci, only Nrv2 isoforms support epithelial SJ barrier function and tracheal tube-size control. Here we show that Nrv1 is endogenously co-expressed with Nrv2 in the epidermis and tracheal system, but Nrv1 has a basolateral localization and appears to be excluded from the Nrv2-containing SJs. When the normally neuronal Nrv3 is expressed in epithelial cells, it does not associate with SJs. Thus, the beta-subunit is a key determinant of Na,K-ATPase subcellular localization as well as function. However, localization of the Na,K-ATPase to SJs is not sufficient for junctional activity because although several Nrv2/Nrv3 chimeric beta-subunits localize to SJs, only those containing the extracellular domain of Nrv2 have junctional activity. Junctional activity is also specific to different alpha-subunit isoforms, with only some isoforms from the major alpha-subunit locus being able to provide full barrier function and produce normal tracheal tubes. Importantly, mutations predicted to inactivate ATPalpha catalytic function do not compromise junctional activity, demonstrating that the Drosophila Na,K-ATPase has an ion-pump-independent role in junction formation and tracheal morphogenesis. These results define new functions for the intensively studied Na,K-ATPase. Strikingly, the rat alpha1 isoform has full junctional activity and can rescue Atpalpha-null mutants to viability, suggesting that the Na,K-ATPase has an evolutionarily conserved role in junction formation and function.