High-capacity Ca2+ binding of human skeletal calsequestrin

Calsequestrin, the major calcium storage protein in both cardiac and skeletal muscle, binds large amounts of Ca(2+) in the sarcoplasmic reticulum and releases them during muscle contraction. For the first time, the crystal structures of Ca(2+) complexes for both human (hCASQ1) and rabbit (rCASQ1) skeletal calsequestrin were determined, clearly defining their Ca(2+) sequestration capabilities through resolution of high- and low-affinity Ca(2+)-binding sites. rCASQ1 crystallized in low CaCl(2) buffer reveals three high-affinity Ca(2+) sites with trigonal bipyramidal, octahedral, and pentagonal bipyramidal coordination geometries, along with three low-affinity Ca(2+) sites. hCASQ1 crystallized in high CaCl(2) shows 15 Ca(2+) ions, including the six Ca(2+) ions in rCASQ1. Most of the low-affinity sites, some of which are μ-carboxylate-bridged, are established by the rotation of dimer interfaces, indicating cooperative Ca(2+) binding that is consistent with our atomic absorption spectroscopic data. On the basis of these findings, we propose a mechanism for the observed in vitro and in vivo dynamic high-capacity and low-affinity Ca(2+)-binding activity of calsequestrin.     

Calsequestrin and the calcium release channel of skeletal and cardiac muscle

Calsequestrin is by far the most abundant Ca(2+)-binding protein in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. It allows the Ca2+ required for contraction to be stored at total concentrations of up to 20mM, while the free Ca2+ concentration remains at approximately 1mM. This storage capacity confers upon muscle the ability to contract frequently with minimal run-down in tension. Calsequestrin is highly acidic, containing up to 50 Ca(2+)-binding sites, which are formed simply by clustering of two or more acidic residues. The Kd for Ca2+ binding is between 1 and 100 microM, depending on the isoform, species and the presence of other cations. Calsequestrin monomers have a molecular mass of approximately 40 kDa and contain approximately 400 residues. The monomer contains three domains each with a compact alpha-helical/beta-sheet thioredoxin fold which is stable in the presence of Ca2+. The protein polymerises when Ca2+ concentrations approach 1mM. The polymer is anchored at one end to ryanodine receptor (RyR) Ca2+ release channels either via the intrinsic membrane proteins triadin and junctin or by binding directly to the RyR. It is becoming clear that calsequestrin has several functions in the lumen of the SR in addition to its well-recognised role as a Ca2+ buffer. Firstly, it is a luminal regulator of RyR activity. When triadin and junctin are present, calsequestrin maximally inhibits the Ca2+ release channel when the free Ca2+ concentration in the SR lumen is 1mM. The inhibition is relieved when the Ca2+ concentration alters, either because of small changes in the conformation of calsequestrin or its dissociation from the junctional face membrane. These changes in calsequestrin's association with the RyR amplify the direct effects of luminal Ca2+ concentration on RyR activity. In addition, calsequestrin activates purified RyRs lacking triadin and junctin. Further roles for calsequestrin are indicated by the kinase activity of the protein, its thioredoxin-like structure and its influence over store operated Ca2+ entry. Clearly, calsequestrin plays a major role in calcium homeostasis that extends well beyond its ability to buffer Ca2+ ions.     

Characterization of cardiac calsequestrin

Calsequestrin, a calcium-binding protein found in the sarcoplasmic reticulum of muscle cells, was purified from rabbit and canine cardiac and skeletal muscle tissue. The amino acid compositions and amino-terminal sequences of skeletal and cardiac calsequestrin from rabbit and dog were determined. The amino acid composition of the cardiac form was very similar to the skeletal form. The amino-terminal sequence of the cardiac form was homologous to, but not identical with, the amino-terminal sequence of the skeletal form of the protein. Few species differences in the amino-terminal sequences were observed. The calcium-binding capacity of the cardiac form was half the capacity of the skeletal form although the affinities of the two forms of calsequestrin for Ca2+ were similar (Kd = 1 mM). Calcium binding to the cardiac form induced structural changes in the protein as determined by circular dichroism and intrinsic fluorescence spectroscopy. The alpha-helical content of cardiac calsequestrin increased from 3.5% to 10.9% upon binding calcium, while the intrinsic fluorescence of the protein increased 14%. Potassium ions also affected the conformation of cardiac calsequestrin.     

High molecular weight calcium binding protein in the microsome of scallop striated muscle

In the microsome of scallop adductor striated muscle, 30K, 55K, 90K, and 360K proteins were detected as calcium binding proteins by 45Ca autoradiography on the transferred nitrocellulose membrane after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The 360K protein was directly extracted with Triton X-100 from the whole homogenate of striated portion of scallop adductor muscle and purified through DEAE cellulose and hydroxyapatite column chromatography. This purified scallop high molecular weight calcium binding protein (SHCBP) showed a faster mobility in SDS PAGE in the presence of Ca2+ than in its absence. The decrease of tryptophan fluorescence had a half maximum near pCa 7 and was slightly co-operative with Mg2+. UV absorbance was slightly increased with Ca2+. The CD spectrum also changed with Mg2+ and Ca2+. These results reflect that this SHCBP binds calcium ions under near physiological conditions. SHCBP-like high molecular weight calcium binding proteins were also detected in the smooth muscle portion of adductor muscle and branchiae of scallop by 45Ca autoradiography, but not in liver. The adductor muscle of clam had a high molecular weight calcium binding protein whose molecular weight was a little smaller than that of SHCBP. The foot of turban shell had the same molecular weight calcium binding protein as SHCBP. Stains-all, a cationic carbocyanine dye, which has been reported to stain calcium binding proteins blue, stained SHCBP blue. The spectrum of SHCBP stained with Stains-all was very similar to that of calsequestrin. Although the function of SHCBP is still unknown, it might be expected to correspond to calsequestrin of vertebrate skeletal muscle, a calcium sequestering protein, in the sarcoplasmic reticulum.     

Polymerization of calsequestrin. Implications for Ca2+ regulation

Two distinct dimerization contacts in calsequestrin crystals suggested a mechanism for Ca(2+) regulation resulting from the occurrence of coupled Ca(2+) binding and protein polymerization. Ca(2+)-induced formation of one contact was proposed to lead to dimerization followed by Ca(2+)-induced formation of the second contact to bring about polymerization (). To test this mechanism, we compared canine cardiac calsequestrin and four truncation mutants with regard to their folding properties, structures, and Ca(2+)-induced polymerization. The wild-type calsequestrin and truncation mutants exhibited similar K(+)-induced folding and end-point structures as indicated by intrinsic fluorescence and circular dichroism, respectively, whereas the polymerization tendencies of the wild-type calsequestrin differed markedly from the polymerization tendencies of the truncation mutants. Static laser light scattering and 3,3'-dithiobis sulfosuccinimidyl-propionate cross-linking indicated that wild-type protein exhibited an initial Ca(2+)-induced dimerization, followed by additional oligomerization as the Ca(2+) concentration was raised or as the K(+) concentration was lowered. None of the truncation mutants exhibited clear stepwise oligomerization that depended on increasing Ca(2+) concentration. Comparison of the three-dimensional structure of rabbit skeletal calsequestrin with a homology model of canine cardiac calsequestrin from the point of view of our coupled Ca(2+) binding and polymerization mechanism leads to a possible explanation for the 2-fold reduced Ca(2+) binding capacity of cardiac calsequestrin despite very similar overall net negative charge for the two proteins.     

Delta 469 mutation in the type 3 repeat calcium binding domain of cartilage oligomeric matrix protein (COMP) disrupts calcium binding

Cartilage oligomeric matrix protein (COMP/TSP5), a large glycoprotein found in the territorial matrix surrounding chondrocytes, is the fifth member of the thrombospondin (TSP) gene family. While the function of COMP is unknown, its importance is underscored by the finding that mutations in the highly conserved type 3 repeat domain causes two skeletal dysplasias. Pseudoachondroplasia (PSACH) and Multiple Epiphyseal Dysplasia, Fairbanks type (EDM1). The type 3 repeats are highly conserved low-affinity Ca(2+)binding domains that are found in all TSP genes. This study was undertaken to determine the effects of mutations on calcium binding and structure of the type 3 repeat domains. Wild-type (WT) and Delta469 recombinant COMP (rCOMP) proteins containing the entire calcium-binding domain were expressed in E. coli and purified. Equilibrium dialysis demonstrated that WT bound 10-12 Ca(2+)ions/molecule while Delta469 bound approximately half the Ca(2+)ions. Circular dichroism (CD) spectrometry had striking spectral changes for the WT in response to increasing concentrations of Ca(2+). These CD spectral changes were cooperative and reversible. In contrast, a large CD spectral change was not observed at any Ca(2+)concentration for Delta469. Moreover, both WT and Delta469 proteins produced similar CD spectral changes when titrated with Zn(2+), Cu(2+)and Ni(2+)indicating that the Delta469 mutation specifically affects only calcium binding. These results suggest that the Delta469 mutation, in the type 3 repeat region, interferes with Ca(2+)binding and that filling of all Ca(2+)binding loops may be critical for correct COMP protein conformation.     

Characterization of high-capacity low-affinity calcium binding protein of liver endoplasmic reticulum: calsequestrin-like and divergent properties

It had been previously demonstrated that endoplasmic reticulum membranes from rat hepatocytes contain a major calsequestrin-like protein, on account of electrophoretic and Stains All-staining properties (Damiani et al., J. Biol. Chem. 263, 340-343). Here we show that a Ca2+-binding protein sharing characteristics in size and biochemical properties with this protein is likewise present in the isolated endoplasmic reticulum from human liver. Human calsequestrin-like protein was characterized as 62 kDa, highly acidic protein (pl 4.5), using an extraction procedure from whole tissue, followed by DEAE-Cellulose chromatography, that was originally developed for purification of skeletal muscle and cardiac calsequestrin. Liver calsequestrin-like protein bound Ca2+ at low affinity (Kd = 4 mM) and in high amounts (Bmax = 1600 nmol Ca2+/mg of protein), as determined by equilibrium dialysis, but differed strikingly from skeletal muscle calsequestrin for the lack of binding to phenyl-Sepharose resin in the absence of Ca2+, and of changes in intrinsic fluorescence upon binding of Ca2+. Thus, these results suggest that liver 62 kDa protein, in spite of its calsequestrin-like Ca2+-binding properties, does not contain a Ca2+-regulated hydrophobic site, which is a specific structural feature of the calsequestrin-class of Ca2+-binding proteins.     

Calsequestrin binds to monomeric and complexed forms of key calcium-handling proteins in native sarcoplasmic reticulum membranes from rabbit skeletal muscle.

Ca(2+)-handling proteins are important regulators of the excitation-contraction-relaxation cycle in skeletal muscle fibres. Although domain binding studies suggest protein coupling between various Ca(2+)-regulatory elements of triad junctions, no direct biochemical evidence exists demonstrating high-molecular-mass complex formation in native microsomal membranes. Calsequestrin represents the protein backbone of the luminal Ca(2+) reservoir and thereby occupies a central position in Ca(2+) homeostasis; we therefore used calsequestrin blot overlay assays in order to determine complex formation between sarcoplasmic reticulum components. Peroxidase-conjugated calsequestrin clearly labelled four major protein bands in one-dimensional (1D) and 2D electrophoretically separated membrane preparations from adult skeletal muscle. Immunoblotting identified the calsequestrin-binding proteins of approximately 26, 63, 94 and 560 kDa as junctin, calsequestrin itself, triadin and the ryanodine receptor, respectively. Protein-protein coupling could be modified by ionic detergents, non-ionic detergents, changes in Ca(2+) concentration, as well as antibody and purified calsequestrin binding. Importantly, complex formation as determined by blot overlay assays was confirmed by differential co-immunoprecipitation experiments and chemical crosslinking analysis. Hence, the key Ca(2+)-regulatory membrane components of skeletal muscle form a supramolecular membrane assembly. The formation of this tightly associated junctional sarcoplasmic reticulum complex seems to underlie the physiological regulation of skeletal muscle contraction and relaxation, which supports the biochemical concept that Ca(2+) homeostasis is regulated by direct protein-protein interactions.     

Effects of thin and thick filament proteins on calcium binding and exchange with cardiac troponin C

Understanding the effects of thin and thick filament proteins on the kinetics of Ca(2+) exchange with cardiac troponin C is essential to elucidating the Ca(2+)-dependent mechanisms controlling cardiac muscle contraction and relaxation. Unlike labeling of the endogenous Cys-84, labeling of cardiac troponin C at a novel engineered Cys-53 with 2-(4'-iodoacetamidoanilo)napthalene-6-sulfonic acid allowed us to accurately measure the rate of calcium dissociation from the regulatory domain of troponin C upon incorporation into the troponin complex. Neither tropomyosin nor actin alone affected the Ca(2+) binding properties of the troponin complex. However, addition of actin-tropomyosin to the troponin complex decreased the Ca(2+) sensitivity ( approximately 7.4-fold) and accelerated the rate of Ca(2+) dissociation from the regulatory domain of troponin C ( approximately 2.5-fold). Subsequent addition of myosin S1 to the reconstituted thin filaments (actin-tropomyosin-troponin) increased the Ca(2+) sensitivity ( approximately 6.2-fold) and decreased the rate of Ca(2+) dissociation from the regulatory domain of troponin C ( approximately 8.1-fold), which was completely reversed by ATP. Consistent with physiological data, replacement of cardiac troponin I with slow skeletal troponin I led to higher Ca(2+) sensitivities and slower Ca(2+) dissociation rates from troponin C in all the systems studied. Thus, both thin and thick filament proteins influence the ability of cardiac troponin C to sense and respond to Ca(2+). These results imply that both cross-bridge kinetics and Ca(2+) dissociation from troponin C work together to modulate the rate of cardiac muscle relaxation.     

Ca2+ binding effects on protein conformation and protein interactions of canine cardiac calsequestrin

Calsequestrin is a Ca2+-binding protein located intraluminally in the junctional sarcoplasmic reticulum (SR) of striated muscle. In this study, Ca2+ binding to cardiac calsequestrin was assessed directly by equilibrium dialysis and correlated with effects on protein conformation and calsequestrin's ability to interact with other SR proteins. Cardiac calsequestrin bound 800-900 nmol of Ca2+/mg of protein (35-40 mol of Ca2+/mol of calsequestrin). Associated with Ca2+ binding to cardiac calsequestrin was a loss in protein hydrophobicity, as revealed with use of absorbance difference spectroscopy, fluorescence emission spectroscopy, and photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125]iodophenyl)diazirine. Ca2+ binding to cardiac calsequestrin also caused a large change in its hydrodynamic character, almost doubling the sedimentation coefficient. We observed that cardiac calsequestrin was very resistant to several proteases after binding Ca2+, consistent with a global effect of Ca2+ on protein conformation. Moreover, Ca2+ binding to cardiac calsequestrin completely prevented its interaction with several calsequestrin-binding proteins, which we identified in cardiac junctional SR vesicles for the first time. The principal calsequestrin-binding protein identified in junctional SR vesicles exhibited an apparent Mr of 26,000 in sodium dodecyl sulfate-polyacrylamide gels. This 26-kDa calsequestrin-binding protein was greatly reduced in free SR vesicles and absent from sarcolemmal vesicles and was different from phospholamban, an SR regulatory protein exhibiting a similar molecular weight. Our results suggest that the specific interaction of calsequestrin with this 26-kDa protein may be regulated by Ca2+ concentration in intact cardiac muscle, when the Ca2+ concentration inside the junctional SR falls to submillimolar levels during coupling of excitation to contraction.     

Calmodulin binding and inhibition of cardiac muscle calcium release channel (ryanodine receptor)

Metabolically (35)S-labeled calmodulin (CaM) was used to determine the CaM binding properties of the cardiac ryanodine receptor (RyR2) and to identify potential channel domains for CaM binding. In addition, regulation of RyR2 by CaM was assessed in [(3)H]ryanodine binding and single-channel measurements. Cardiac sarcoplasmic reticulum vesicles bound approximately four CaM molecules per RyR2 tetramer in the absence of Ca(2+); in the presence of 100 microm Ca(2+), the vesicles bound 7.5 CaM molecules per tetramer. Purified RyR2 bound approximately four [(35)S]CaM molecules per RyR tetramer, both in the presence and absence of Ca(2+). At least four CaM binding domains were identified in [(35)S]CaM overlays of fusion proteins spanning the full-length RyR2. The affinity (but not the stoichiometry) of CaM binding was altered by redox state as controlled by the presence of either GSH or GSSG. Inhibition of RyR2 activity by CaM was influenced by Ca(2+) concentration, redox state, and other channel modulators. Parallel experiments with the skeletal muscle isoform showed major differences in the CaM binding properties and regulation by CaM of the skeletal and cardiac ryanodine receptors.     

Subproteomics analysis of Ca+-binding proteins demonstrates decreased calsequestrin expression in dystrophic mouse skeletal muscle.

Duchenne muscular dystrophy represents one of the most common hereditary diseases. Abnormal ion handling is believed to render dystrophin-deficient muscle fibres more susceptible to necrosis. Although a reduced Ca(2+) buffering capacity has been shown to exist in the dystrophic sarcoplasmic reticulum, surprisingly no changes in the abundance of the main luminal Ca(2+) reservoir protein calsequestrin have been observed in microsomal preparations. To address this unexpected finding and eliminate potential technical artefacts of subcellular fractionation protocols, we employed a comparative subproteomics approach with total mouse skeletal muscle extracts. Immunoblotting, mass spectrometry and labelling of the entire muscle protein complement with the cationic carbocyanine dye 'Stains-All' was performed in order to evaluate the fate of major Ca(2+)-binding proteins in dystrophin-deficient skeletal muscle fibres. In contrast to a relatively comparable expression pattern of the main protein population in normal vs. dystrophic fibres, our analysis showed that the expression of key Ca(2+)-binding proteins of the luminal sarcoplasmic reticulum is drastically reduced. This included the main terminal cisternae constituent, calsequestrin, and the previously implicated Ca(2+)-shuttle element, sarcalumenin. In contrast, the 'Stains-All'-positive protein spot, representing the cytosolic Ca(2+)-binding component, calmodulin, was not changed in dystrophin-deficient fibres. The reduced 2D 'Stains-All' pattern of luminal Ca(2+)-binding proteins in mdx preparations supports the calcium hypothesis of muscular dystrophy. The previously described impaired Ca(2+) buffering capacity of the dystrophic sarcoplasmic reticulum is probably caused by a reduction in luminal Ca(2+)-binding proteins, including calsequestrin.     

Mutation of the high affinity calcium binding sites in cardiac troponin C.

Fast skeletal and cardiac troponin C (TnC) contain two high affinity Ca2+/Mg2+ binding sites within the C-terminal domain that are thought to be important for association of TnC with the troponin complex of the thin filament. To test directly the function of these high affinity sites in cardiac TnC they were systematically altered by mutagenesis to generate proteins with a single inactive site III or IV (CBM-III and CBM-IV, respectively), or with both sites III and IV inactive (CBM-III-IV). Equilibrium dialysis indicated that the mutated sites did not bind Ca2+ at pCa 4. Both CBM-III and CBM-IV were similar to the wild type protein in their ability to regulate Ca(2+)-dependent contraction in slow skeletal muscle fibers, and Ca(2+)-dependent ATPase activity in fast skeletal and cardiac muscle myofibrils. The mutant CBM-III-IV is capable of regulating contraction in permeabilized slow muscle fibers but only if the fibers are maintained in a contraction solution containing a high concentration of the mutant protein. CBM-III-IV also regulates myofibril ATPase activity in fast skeletal and cardiac myofibrils but only at concentrations 10-100-fold greater than the normal protein. The pCa50 and Hill coefficient values for Ca(2+)-dependent activation of fast skeletal muscle myofibril ATPase activity by the normal protein and all three mutants are essentially the same. Competition between active and inactive forms of cardiac and slow TnC in a functional assay demonstrates that mutation of both sites III and IV greatly reduces the affinity of cardiac and slow TnC for its functionally relevant binding site in the myofibrils. The data indicate that although neither high affinity site is absolutely essential for regulation of muscle contraction in vitro, at least one active C-terminal site is required for tight association of cardiac troponin C with myofibrils. This requirement can be satisfied by either site III or IV.     

Interaction of HRC (histidine-rich Ca(2+)-binding protein) and triadin in the lumen of sarcoplasmic reticulum

HRC (histidine-rich Ca(2+) binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca(2+) with high capacity and low affinity. While HRC resides in the lumen of the sarcoplasmic reticulum, the physiological function of HRC is largely unknown. In the present study, we have performed co-immunoprecipitation experiments and show that HRC binds directly to triadin, which is an integral membrane protein of the sarcoplasmic reticulum. Using a fusion protein binding assay, we further identified the histidine-rich acidic repeats of HRC as responsible for the binding of HRC to triadin. These motifs may represent a novel protein-protein interaction domain. The HRC binding domain of triadin was also localized by fusion protein binding assay to the lumenal region containing the KEKE motif that was previously shown to be involved in the binding of triadin to calsequestrin. Notably, the interaction of HRC and triadin is Ca(2+)-sensitive. Our data suggest that HRC may play a role in the regulation of Ca(2+) release from the sarcoplasmic reticulum by interaction with triadin.     

Low-affinity Ca(2+)-binding sites versus Zn(2+)-binding sites in histidine-rich Ca(2+)-binding protein of skeletal muscle sarcoplasmic reticulum.

Histidine-rich Ca(2+)-binding protein (HRC) is a 170 kDa protein that can be identified in the isolated sarcoplasmic reticulum from rabbit skeletal muscle by its ability to bind [125I]low-density lipoprotein on blots after SDS-PAGE and that appears to be bound to the junctional membrane through calcium bridges. Molecular cDNA cloning of this protein predicts the existence of a Ca(2+)-binding domain and of a distinct heavy-metal binding domain at the cystein-rich COOH-terminus. Here we demonstrate, using radioactive ligand blot techniques, that HRC protein binds 45Ca at low affinity, as well as being able to bind 65Zn, but at different sites, that are largely inhibitable by prior reductive alkylation of the protein. In contrast to Ca(2+)-binding protein calsequestrin not having detectable 65Zn-binding sites, HRC protein bound selectively to immobilized Zn2+ on IDA-agarose affinity columns. Our results also indicate that rabbit and human 140 kDa HRC protein have common properties.     

Determinants for calmodulin binding on voltage-dependent Ca2+ channels

Calmodulin, bound to the alpha(1) subunit of the cardiac L-type calcium channel, is required for calcium-dependent inactivation of this channel. Several laboratories have suggested that the site of interaction of calmodulin with the channel is an IQ-like motif in the carboxyl-terminal region of the alpha(1) subunit. Mutations in this IQ motif are linked to L-type Ca(2+) current (I(Ca)) facilitation and inactivation. IQ peptides from L, P/Q, N, and R channels all bind Ca(2+)calmodulin but not Ca(2+)-free calmodulin. Another peptide representing a carboxyl-terminal sequence found only in L-type channels (designated the CB domain) binds Ca(2+)calmodulin and enhances Ca(2+)-dependent I(Ca) facilitation in cardiac myocytes, suggesting the CB domain is functionally important. Calmodulin blocks the binding of an antibody specific for the CB sequence to the skeletal muscle L-type Ca(2+) channel, suggesting that this is a calmodulin binding site on the intact protein. The binding of the IQ and CB peptides to calmodulin appears to be competitive, signifying that the two sequences represent either independent or alternative binding sites for calmodulin rather than both sequences contributing to a single binding site.     

The calcium binding properties of phosphorylated and unphosphorylated cardiac and skeletal myosins.

Porcine left ventricular cardiac myosin and rabbit white skeletal myosin were phosphorylated by rabbit skeletal myosin light chain kinase and their Ca2+ binding properties were examined by equilibrium dialysis techniques. No significant effect of phosphorylation on the Ca2+ binding properties of these myosins was observed. Both types of striated muscle myosins bound approximately 2 mol of Ca2+/mol of myosin with similar affinities of 3 x 10(7) M-1. In the presence of 3 x 10(-4) M Mg2+ the myosins bound Ca2+ with a reduced affinity of 3 to 4 x 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the binding sites on myosin, the changes in Ca2+ binding can be accounted for by a Mg2+ affinity of 2.5 to 3.0 x 10(5) M-1.     

Calsequestrin distribution, structure and function, its role in normal and pathological situations and the effect of thyroid hormones

Calsequestrin is the main calcium binding protein of the sarcoplasmic reticulum, serving as an important regulator of Ca(2+). In mammalian muscles, it exists as a skeletal isoform found in fast- and slow-twitch skeletal muscles and a cardiac isoform expressed in the heart and slow-twitch muscles. Recently, many excellent reviews that summarised in great detail various aspects of the calsequestrin structure, localisation or function both in skeletal and cardiac muscle have appeared. The present review focuses on skeletal muscle: information on cardiac tissue is given, where differences between both tissues are functionally important. The article reviews the known multiple roles of calsequestrin including pathology in order to introduce this topic to the broader scientific community and to stimulate an interest in this protein. Newly we describe our results on the effect of thyroid hormones on skeletal and cardiac calsequestrin expression and discuss them in the context of available literary data on this topic.     

Phosphorylation of the cardiac isoform of calsequestrin in cultured rat myotubes and rat skeletal muscle.

Calsequestrin is a high-capacity Ca(2+)-binding protein and a major constituent of the sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle. Two isoforms of calsequestrin, cardiac and skeletal muscle forms, have been described which are products of separate genes. Purified forms of the two prototypical calsequestrin isoforms, dog cardiac and rabbit fast-twitch skeletal muscle calsequestrins, serve as excellent substrates for casein kinase II and are phosphorylated on distinct sites (Cala, S.E. and Jones, L.R. (1991) J. Biol. Chem 266, 391-398). Dog cardiac calsequestrin is phosphorylated at a 50 to 100-fold greater rate than is rabbit skeletal muscle calsequestrin, and only the dog cardiac isoform contains endogenous Pi on casein kinase II phosphorylation sites. In this study, we identified and examined both calsequestrin isoforms in rat muscle cultures and homogenates to demonstrate that the cardiac isoform of calsequestrin in rat skeletal muscle was phosphorylated in vivo on sites which are phosphorylated by casein kinase II in vitro. Phosphorylation of rat skeletal muscle calsequestrin was not detected. In tissue homogenates, cardiac and skeletal muscle calsequestrin isoforms were both found to be prominent substrates for endogenous casein kinase II activity with cardiac calsequestrin the preferred substrate. In addition, these studies revealed that the cardiac isoform of calsequestrin was the predominant form expressed in skeletal muscle of fetal rats and cultured myotubes.     

Thermodynamics of calmodulin binding to cardiac and skeletal muscle ryanodine receptor ion channels

The skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptor calcium release channels contain a single, conserved calmodulin (CaM) binding domain, yet are differentially regulated by CaM. Here, we report that high-affinity [(35)S]CaM binding to RyR1 is driven by favorable enthalpic and entropic contributions at Ca(2+) concentrations from <0.01 to 100 microM. At 0.15 microM Ca(2+), [(35)S]CaM bound to RyR2 with decreased affinity and binding enthalpy compared with RyR1. The rates of [(35)S]CaM dissociation from RyR1 increased as the temperature was raised, whereas at 0.15 microM Ca(2+) the rate from RyR2 was little affected. The results suggest major differences in the energetics of CaM binding to and dissociation from RyR1 and RyR2.