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.     

Isoform-specific functions of Na,K-ATPase in skeletal muscle

The published data and the results of the author’s own research in the field of the molecular and functional diversity of Na,K-ATPases are reviewed. Na,K-ATPase is an integral membrane protein that maintains the concentration gradients of Na⁺ and K⁺ that are essential for electrogenesis, excitability, and several other processes of cellular transport. Most of the Na,K-ATPase of vertebrates is found in the skeletal muscle tissue, which co-expresses the α1 and α2 isoforms of the catalytic and transport α-subunit of Na,KATPase. The activity of Na,K-ATPase is crucial for the contractile function and prolonged activity of skeletal muscle. The data that have accumulated indicate that the α1 isoform of Na,K-ATPase fulfills the major pumping function. The α2 isoform fulfills additional functions related to the specific membrane localization of the protein, the functional interactions with the proteins and lipids of the environment, and fine-tuned regulation by a variety of factors, including motor activity.     

Identification of a novel tropomodulin isoform, skeletal tropomodulin, that caps actin filament pointed ends in fast skeletal muscle.

Tropomodulin (E-Tmod) is an actin filament pointed end capping protein that maintains the length of the sarcomeric actin filaments in striated muscle. Here, we describe the identification and characterization of a novel tropomodulin isoform, skeletal tropomodulin (Sk-Tmod) from chickens. Sk-Tmod is 62% identical in amino acid sequence to the previously described chicken E-Tmod and is the product of a different gene. Sk-Tmod isoform sequences are highly conserved across vertebrates and constitute an independent group in the tropomodulin family. In vitro, chicken Sk-Tmod caps actin and tropomyosin-actin filament pointed ends to the same extent as does chicken E-Tmod. However, E- and Sk-Tmods differ in their tissue distribution; Sk-Tmod predominates in fast skeletal muscle fibers, lens, and erythrocytes, while E-Tmod is found in heart and slow skeletal muscle fibers. Additionally, their expression is developmentally regulated during chicken breast muscle differentiation with Sk-Tmod replacing E-Tmod after hatching. Finally, in skeletal muscle fibers that coexpress both Sk- and E-Tmod, they are recruited to different actin filament-containing cytoskeletal structures within the cell: myofibrils and costameres, respectively. All together, these observations support the hypothesis that vertebrates have acquired different tropomodulin isoforms that play distinct roles in vivo.     

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.     

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.     

alphaKAP is an anchoring protein for a novel CaM kinase II isoform in skeletal muscle.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is present in a membrane-bound form that phosphorylates synapsin I on neuronal synaptic vesicles and the ryanodine receptor at skeletal muscle sarcoplasmic reticulum (SR), but it is unclear how this soluble enzyme is targeted to membranes. We demonstrate that alphaKAP, a non-kinase protein encoded by a gene within the gene of alpha-CaM kinase II, can target the CaM kinase II holoenzyme to the SR membrane. Our results indicate that alphaKAP (i) is anchored to the membrane via its N-terminal hydrophobic domain, (ii) can co-assemble with catalytically competent CaM kinase II isoforms and target them to the membrane regardless of their state of activation, and (iii) is co-localized and associated with rat skeletal muscle CaM kinase II in vivo. alphaKAP is therefore the first demonstrated anchoring protein for CaM kinase II. CaM kinase II assembled with alphaKAP retains normal enzymatic activity and the ability to become Ca2+-independent following autophosphorylation. A new variant of beta-CaM kinase II, termed betaM-CaM kinase II, is one of the predominant CaM kinase II isoforms associated with alphaKAP in skeletal muscle SR.     

cDNA cloning of the beta-subunit of the human gastric H,K-ATPase.

A full-length cDNA clone encoding the human gastric H,K-ATPase (EC 3.6.1.36)beta-subunit was isolated from a human gastric mucosal lambda gt10 library using oligonucleotide probes which were based on the cDNA sequence from rat and rabbit H,K-ATPase beta-subunits. The insert was 1407 bp in length and encoded a polypeptide of 291 amino acids with a MW = 33,367 Da. It exhibited 84.2%, 85.6% and 81.3% identity to the H,K-ATPase beta-subunits of rabbit, pig and rat, respectively.     

The H,K-ATPase beta-subunit can act as a surrogate for the beta-subunit of Na,K-pumps.

Na,K-ATPase and H,K-ATPase are the only members of the P-type ATPases in which a glycosylated beta-subunit is part of the purified active enzyme. In this study, we have followed the synthesis and the posttranslational processing of the beta-subunit of H,K-ATPase (beta HK) in Xenopus oocytes injected with beta HK cRNA and have tested whether it can act as a surrogate for the beta-subunit of Na,K-ATPase (beta NaK) to support the functional expression of Na,K-pumps. In Xenopus oocytes, beta HK is processed from an Endo H-sensitive 51-kDa coreglycosylated form to an Endo H-resistant 71-kDa fully glycosylated form. Similar to beta NaK, beta HK can stabilize and increase the trypsin resistance of alpha-subunits of Na,K-ATPase (alpha NaK). Finally, expression of beta HK together with alpha NaK leads to an increased number of ouabain binding sites at the plasma membrane accompanied by an increased Rb+ uptake and Na,K-pump current. Our data suggest that beta HK, similar to beta NaK, can assemble to alpha NaK, support the structural maturation and the intracellular transport of catalytic alpha NaK, and ultimately form active alpha NaK-beta HK complexes with Na,K-pump transport properties.     

Accumulation of beta (m), a structural member of X,K-ATPase beta-subunit family, in nuclear envelopes of perinatal myocytes

Recently discovered muscle-specific beta(m) protein is structurally closely related to the X,K-ATPase beta-subunits. However, it has a number of unique properties such as predominant localization in intracellular stores and lack of association with known X,K-ATPase alpha-subunits on heterologous coexpression. In this study, the primary structure of mouse beta(m) was determined and developmental regulation of the gene (ATP1B4) was analyzed. The expression is first detected at day 14 of gestation, is sharply increased at day 16, and reaches its maximum at day 18. After birth, the expression quickly decreases and is hardly detectable in adult mice. A more detailed subcellular localization study was undertaken, and its results indicate that beta(m) not only is located in sarcoplasmic reticulum but is concentrated in nuclear envelopes of both prenatal and postnatal skeletal muscles. Immunohistochemical studies show that beta(m) is specific to myocytes and, at the subcellular level, many nuclear envelopes are intensively labeled in both fetal and newborn skeletal muscles. Accordingly, beta(m) is detected by immunoblotting in purified nuclei and nuclear membranes from neonatal skeletal muscles. On transfection of human rhabdomyosarcoma cell line RD, green fluorescent protein-tagged beta(m) resides intracellularly with significant enrichment in nuclear envelopes, whereas beta(m) with transmembrane domain deleted localizes in both cytoplasm and nucleoplasm. Nuclear beta(m) apparently is not in association with Na,K-ATPase because we never detected its alpha-subunit in myonuclear membranes. These results indicate that beta(m) has a specialized function in mammalian perinatal myocytes, different from functions of other X,K-ATPase beta-subunits. The unique temporospatial distribution of beta(m) protein expression suggests its important role in development of growing skeletal muscle.     

Immunochemical analysis of the peroxisomal beta-oxidation enzymes in rat and human heart and skeletal muscle and in skeletal muscle of Zellweger patients

Immunoblot analyses with antibodies against the peroxisomal beta-oxidation enzymes from rat liver showed the presence of these enzymes in rat and human liver and kidney and rat adrenal gland. The bifunctional protein could not be detected in muscle tissues or cultured muscle cells. Acyl-CoA oxidase was detected in rat heart and cultured human muscle cells. 3-Ketoacyl-CoA thiolase was also detected in human and rat heart and skeletal muscle; however, this enzyme was not detectable in skeletal muscle of Zellweger patients, in agreement with the absence of peroxisomal fatty acid oxidation.     

Monoclonal antibody ALD 180: a reagent specific for a human prenatal skeletal muscle myosin isoform

We report the characterization of monoclonal antibody (MAb) ALD 180, prepared against the myosin of slow avian muscle, for studies of human muscle development and disease. With the use of radioimmunoassays, Western immunoblots of native and denatured myosins, and epifluorescent indirect immunocytochemistry, we show that ALD 180 is specific for an epitope in human prenatal skeletal muscle myosin heavy chain (MHC), which is expressed in diminishing abundance in fetal fibers from at least 19-22 weeks' gestation to term and also in regenerating muscle fibers seen in diseased muscles from both children and adults. ALD 180 recognizes an epitope apparently different from those reacting with anti-prenatal human myosin MAb previously described, and therefore affords a complementary reagent for use in future studies of human myosin isoform expression and regulation.     

Cloning and characterization of a new isoform of skeletal muscle triadin

We have shown that several isoforms of triadin, a protein involved in calcium release process through the ryanodine receptor, are expressed in rat skeletal muscle, and we have cloned two of these isoforms. One is the rat homolog of the 95-kDa triadin identified in rabbit skeletal muscle, and the second one, shorter, is a truncated form of the previous one, but with a new unique COOH-terminal end. We propose to name the two proteins identified here Trisk 95 and Trisk 51. We have produced antibodies specific to each isoform. Using these antibodies, we have shown that the newly identified protein, Trisk 51, is actually expressed in adult rat skeletal muscle and also in rat embryo skeletal muscle. Immunofluorescent labeling of rat skeletal muscle with anti-Trisk 95, anti-Trisk 51, or anti-ryanodine receptor antibodies shows a similar localization of these proteins, in the tissue. Transfection of L6 cells with cDNA of Trisk 51 or Trisk 95 leads to the expression of proteins with the expected molecular weight, identical to those detected in rat skeletal muscle. Both proteins appear during differentiation of satellite cells in myotubes which may indicate the involvement of these two isoforms in the building of a functional calcium release machinery.     

Immunohistochemical demonstration of fibre type-specific isozymes of cytochrome c oxidase in human skeletal muscle

Summary The immunohistochemical reaction of monoclonal as well as polyclonal antibodies against cytochrome c oxidase (COX) subunits with serial sections of normal human skeletal muscle was investigated. The stronger reactivity of polyclonal antibodies to COX subunits II–III and VIIbc with type I as compared to type II fibres, correlated well with the higher histochemical reactivity of NADH dehydrogenase, succinate dehydrogenase and cytochrome c oxidase in type I fibres. In contrast an almost exclusive reaction of a monoclonal antibody against subunit IV with type I fibre and a preponderan reaction of a polyclonal antibody against subunits Vab with type II fibres was obtained. Antibodies against subuntis I, Vb and VIc did not reveal a fibre-type-specific reactivity. The data indicate in human muscle the occurrence of fibre type-specific isozymes of cytochrome c oxidase differing in subunits IV and Va or Vb.