Drastic reduction in the luminal Ca2+ -binding proteins calsequestrin and sarcalumenin in dystrophin-deficient cardiac muscle

Luminal Ca2+ -binding proteins play a central role in mediating between Ca2+ -uptake and Ca2+ -release during the excitation-contraction-relaxation cycle in muscle fibres. In the most commonly inherited neuromuscular disorder, Duchenne muscular dystrophy (DMD), the reduced expression of key Ca2+ -binding proteins causes abnormal Ca2+ -buffering in the sarcoplasmic reticulum (SR) of skeletal muscle. The heart is also affected in dystrophinopathies, as manifested by the pathological replacement of cardiac fibres by connective and fatty tissue. We therefore investigated whether similar changes occur in the abundance of luminal Ca2+ -regulatory elements in dystrophin-deficient cardiac fibres. Two-dimensional immunoblotting of total cardiac extracts was employed to unequivocally determine potential changes in the expression levels of SR components. Interestingly, the expression of the histidine-rich Ca2+ -binding protein was increased in the dystrophic heart. In contrast, the major Ca2+ -reservoir protein of the terminal cisternae, calsequestrin (CSQ), and the Ca2+ -shuttle and ion-binding protein of the longitudinal tubules, sarcalumenin, were drastically reduced in cardiac mdx fibres. This result agrees with the recently reported decrease in the Ca2+ -release channel and Ca2+ -ATPase in the mdx heart. Abnormal Ca2+ -handling appears to play a major role in the molecular pathogenesis of the cardiac involvement in X-linked muscular dystrophy.     

Comparative analysis of Dp427-deficient mdx tissues shows that the milder dystrophic phenotype of extraocular and toe muscle fibres is associated with a persistent expression of beta-dystroglycan

The cell biological hypothesis of Duchenne muscular dystrophy assumes that deficiency in the membrane cytoskeletal element dystrophin triggers a loss in surface glycoproteins, such as beta-dystroglycan, thereby rendering the sarcolemmal membrane more susceptible to micro-rupturing. Secondary changes in ion homeostasis, such as increased cytosolic Ca2+ levels and impaired luminal Ca2+ buffering, eventually lead to Ca2+-induced myonecrosis. However, individual muscle groups exhibit a graded pathological response during the natural time course of x-linked muscular dystrophy. The absence of the dystrophin isofom Dp427 does not necessarily result in a severe dystrophic phenotype in all muscle groups. In the dystrophic mdx animal model, extraocular and toe muscles are not as severely affected as limb muscles. Here, we show that the relative expression and sarcolemmal localization of the central trans-sarcolemmal linker of the dystrophin-glycoprotein complex, beta-dystroglycan, is preserved in mdx extraocular and toe fibres by means of two-dimensional immunoblotting and immunofluorescence microscopy. Thus, with respect to improving myology diagnostics, the relative expression levels of beta-dystroglycan appear to represent reliable markers for the severity of secondary changes in dystrophin-deficient fibres. Immunoblotting and enzyme assays revealed that mdx toe muscle fibres exhibit an increased expression and activity of the sarcoplasmic reticulum Ca2+-ATPase. Chemical crosslinking studies demonstrated impaired calsequestrin oligomerization in mdx gastrocnemius muscle indicating that abnormal calsequestrin clustering is involved in reduced Ca2+ buffering of the dystrophic sarcoplasmic reticulum. Previous studies have mostly attributed the sparing of certain mdx fibres to the special protective properties of small-diameter fibres. Our study suggests that the rescue of dystrophin-associated glycoproteins, and possibly the increased removal of cytosolic Ca2+ ions, might also play an important role in protecting muscle cells from necrotic changes.     

Reduced expression of regucalcin in young and aged mdx diaphragm indicates abnormal cytosolic calcium handling in dystrophin-deficient muscle

The cytosolic Ca2+ -binding protein regucalcin is involved in intracellular signaling and present in high abundance in the liver. Here, we could show by comparative mass spectrometry-based proteomics screening of normal versus dystrophic fibres that regucalcin of 33.9 kDa and pI5.2 also exists in diaphragm muscle. Since the expression of sarcolemmal Ca2+ -leak channels and luminal Ca2+ -binding elements is altered in dystrophin-deficient muscle, we initiated this study in order to determine whether additional soluble muscle proteins involved in Ca2+ -handling are affected in muscular dystrophy. Following separation by two-dimensional gel electrophoresis, the spot pattern of the normal versus the mdx diaphragm muscle proteome was evaluated by densitometry. The expression levels of 20 major protein spots were shown to change and their identity determined by mass spectrometry. A 2-fold reduction of regucalcin in mdx diaphragm, as well as in dystrophic limb muscle and heart, was confirmed by immunoblotting in both young and aged mdx mice. The results from our proteomics analysis of dystrophic diaphragm support the concept that abnormal Ca2+ -handling is involved in x-linked muscular dystrophy. The reduction in key Ca2+ -handling proteins may result in an insufficient maintenance of Ca2+ -homeostasis and an abnormal regulation of Ca2+ -dependent enzymes resulting in disturbed intracellular signaling mechanisms in dystrophinopathies.     

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.     

Drastic reduction of calsequestrin-like proteins and impaired calcium binding in dystrophic mdx muscle.

Although the reduction in dystrophin-associated glycoproteins is the primary pathophysiological consequence of the deficiency in dystrophin, little is known about the secondary abnormalities leading to x-linked muscular dystrophy. As abnormal Ca(2+) handling may be involved in myonecrosis, we investigated the fate of key Ca(2+) regulatory membrane proteins in dystrophic mdx skeletal muscle membranes. Whereas the expression of the ryanodine receptor, the dihydropyridine receptor, the Ca(2+)-ATPase, and calsequestrin was not affected, a drastic decline in calsequestrin-like proteins of 150-220 kDa was observed in dystrophic microsomes using one-dimensional immunoblotting, two-dimensional immunoblotting with isoelectric focusing, diagonal two-dimensional blotting technique, and immunoprecipitation. In analogy, overall Ca(2+) binding was reduced in the sarcoplasmic reticulum of dystrophic muscle. The reduction in Ca(2+) binding proteins might be directly involved in triggering impaired Ca(2+) sequestration within the lumen of the sarcoplasmic reticulum. Thus disturbed sarcolemmal Ca(2+) fluxes seem to influence overall Ca(2+) homeostasis, resulting in distinct changes in the expression profile of a subset of Ca(2+) handling proteins, which might be an important factor in the progressive functional decline of dystrophic muscle fibers.     

Analysis of excitation-contraction-coupling components in chronically stimulated canine skeletal muscle.

The chronic stimulation of predominantly fast-twitch mammalian skeletal muscle causes a transformation to physiological characteristics of slow-twitch skeletal muscle. Here, we report the effects of chronic stimulation on the protein components of the sarcoplasmic reticulum and transverse tubular membranes which are directly involved in excitation-contraction coupling. Comparison of protein composition of microsomal fractions from control and chronically stimulated muscle was performed by immunoblot analysis and also by staining with Coomassie blue or the cationic carbocyanine dye Stains-all. Consistent with previous experiments, a greatly reduced density was observed for the fast-twitch isozyme of Ca(2+)-ATPase, while the expression of the slow-twitch Ca(2+)-ATPase was found to be greatly enhanced. Components of the sarcolemma (Na+/K(+)-ATPase, dystrophin-glycoprotein complex) and the free sarcoplasmic reticulum (Ca(2+)-binding protein sarcalumenin and a 53-kDa glycoprotein) were not affected by chronic stimulation. The relative abundance of calsequestrin was slightly reduced in transformed skeletal muscle. However, the expression of the ryanodine receptor/Ca(Ca2+)-release channel from junctional sarcoplasmic reticulum and the transverse tubular dihydropyridine-sensitive Ca2+ channel, as well as two junctional sarcoplasmic reticulum proteins of 90 kDa and 94 kDa, was greatly suppressed in transformed muscle. Thus, the expression of the major protein components of the triad junction involved in excitation-contraction coupling is suppressed, while the expression of other muscle membrane proteins is not affected in chronically stimulated muscle.     

Comparison of the (Ca2+ + Mg2+)-ATPase proteins from normal and dystrophic chicken sarcoplasmic reticulum.

The involvement of membrane protein in dystrophic chicken fragmented sarcoplasmic reticulum alterations has been examined. A purified preparation of the (Ca2+ + Mg2+)-ATPase protein from dystrophic fragmented sarcoplasmic reticulum was found to have a reduced calcium-sensitive ATPase activity and phosphoenzyme level, in agreement with alterations found in dystrophic chicken fragmented sarcoplasmic reticulum. An amino acid analysis of the ATPase preparations showed no difference in the normal and dystrophic (Ca2+ + Mg2+)-ATPase. The (Ca2+ + Mg2+)-ATPase was investigated further by isoelectric focusing and proteolytic digestion of the fragmented sarcoplasmic reticulum. Neither of these methods indicated any alteration in the composition of the dystrophic (Ca2+ + Mg2+)-ATPase. We have concluded that the alterations observed in dystrophic fragmented sarcoplasmic reticulum are not due to increased amounts of non-(Ca2+ + Mg2+)-ATPase protein, and that the normal and dystrophic (Ca2+ + Mg2+)-ATPase protein are not detectably different.     

Calreticulin is a candidate for a calsequestrin-like function in Ca2(+)-storage compartments (calciosomes) of liver and brain.

In a search for the non-muscle equivalent of calsequestrin (the low-affinity high-capacity Ca2(+)-binding protein responsible for Ca2+ storage within the terminal cisternae of the sarcoplasmic reticulum), acidic proteins were extracted from rat liver and brain microsomal preparations and purified by column chromatography. No calsequestrin was observed in these extracts, but the N-terminal amino acid sequence of the major Ca2(+)-binding protein of the liver microsomal fraction was determined and found to correspond to that of calreticulin. This protein was found to bind approx. 50 mol of Ca2+/mol of protein, with low affinity (average Kd approx. 1.0 mM). A monoclonal antibody, C6, raised against skeletal-muscle calsequestrin cross-reacted with calreticulin in SDS/PAGE immunoblots, but polyclonal antibodies reacted with native calreticulin only weakly, or not at all, after SDS denaturation. Immuno-gold decoration of liver ultrathin cryosections with affinity-purified antibodies against liver calreticulin revealed luminal labelling of vacuolar profiles indistinguishable from calciosomes, the subcellular structures previously identified by the use of anti-calsequestrin antibodies. We conclude that calreticulin is the Ca2(+)-binding protein segregated within the calciosome lumen, previously described as being calsequestrin-like. Because of its properties and intraluminal location, calreticulin might play a critical role in Ca2+ storage and release in non-muscle cells, similar to that played by calsequestrin in the muscle sarcoplasmic reticulum.     

Postnatal development of Ca2+-sequestration by the sarcoplasmic reticulum of fast and slow muscles in normal and dystrophic mice

Ca2+-uptake activities of the sarcoplasmic reticulum (SR) were determined with a Ca2+-sensitive electrode in homogenates from fast- and slow-twitch muscles from both normal and dystrophic mice (C57BL/6J strain) of different ages. Immunochemical quantification of tissue Ca2+-ATPase content allowed determination of the specific Ca2+-transport activity of the enzyme. In 3-week-old mice of the dystrophic strain specific Ca2+ transport was already significantly lower than in the normal strain. It progressively decreased with maturation and reached only 40-50% and 30-50% of the normal values in fast- and slow-twitch muscles of adult dystrophic animals, respectively. Tissue contents of calsequestrin were reduced in both types of muscle leading to an increased Ca2+-ATPase to calsequestrin protein ratio. Equal amounts of the Ca2+-ATPase protein (detected by Coomassie blue staining of polyacrylamide gels) were present in SR vesicles isolated by Ca2+-oxalate loading from adult normal and dystrophic fast-twitch muscles. However, the specific ATP-hydrolysing activity of the enzyme was approximately 50% lower in dystrophic than in normal SR. The reduced ATP-hydrolysing activity was correlated with decreased Ca2+-transport activity, phosphoprotein formation and fluorescein isothiocyanate labeling as determined in total microsomal and heavy SR fractions. Although the Ca2+ and ATP affinities of the enzyme were unaltered, its ATPase activity was reduced at all levels of ATP in the dystrophic SR. Taken together, these findings point to a markedly impaired function of the SR and an increase in the population of inactive SR Ca2+-ATPase molecules in murine muscular dystrophy.     

Rapid purification of calsequestrin from cardiac and skeletal muscle sarcoplasmic reticulum vesicles by Ca2+-dependent elution from phenyl-sepharose

Treatment of cardiac or skeletal muscle sarcoplasmic reticulum vesicles with 0.1 M sodium carbonate selectively extracts both the Ca2+-binding protein calsequestrin and the two "intrinsic glycoproteins," while leaving the Ca2+-dependent ATPase membrane bound. Phenyl-Sepharose chromatography in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and high salt (0.5 M NaCl) readily fractionates these solubilized proteins into a Ca2+-elutable fraction, which contains purified calsequestrin, and a low ionic strength elutable fraction, which contains one of the two intrinsic glycoproteins. Elution of calsequestrin from phenyl-Sepharose occurs near 1 mM Ca2+. Copurifying with calsequestrin are an homologous set of high molecular weight proteins, which like calsequestrin stain blue with Stains-All. These proteins are present in trace amounts and do not correspond to any sarcoplasmic reticulum proteins previously identified. Elution of calsequestrin from phenyl-Sepharose is consistent with the Ca2+-binding protein losing its hydrophobic character in the presence of millimolar Ca2+. This behavior is converse to that observed for several calmodulin-like proteins, which are eluted from hydrophobic gels in the presence of EGTA. The high yield and purity of calsequestrin prepared by this method makes possible a unique system for studying what may be a distinct class of Ca2+-binding proteins.     

Interaction of ruthenium red with Ca2(+)-binding proteins.

The interaction of ruthenium red, [(NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5]Cl6.4H2O, with various Ca2(+)-binding proteins was studied. Ruthenium red inhibited Ca2+ binding to the sarcoplasmic reticulum protein, calsequestrin, immobilized on Sepharose 4B. Furthermore, ruthenium red bound to calsequestrin with high affinity (Kd = 0.7 microM; Bmax = 218 nmol/mg protein). The dye stained calsequestrin in sodium dodecyl sulfate-polyacrylamide gels or on nitrocellulose paper and was displaced by Ca2+ (Ki = 1.4 mM). The specificity of ruthenium red staining of several Ca2(+)-binding proteins was investigated by comparison with two other detection methods, 45Ca2+ autoradiography and the Stains-all reaction. Ruthenium red bound to the same proteins detected by the 45Ca2+ overlay technique. Ruthenium red stained both the erythrocyte Band 3 anion transporter and the Ca2(+)-ATPase of skeletal muscle sarcoplasmic reticulum. Ruthenium red also stained the EF hand conformation Ca2(+)-binding proteins, calmodulin, troponin C, and S-100. This inorganic dye provides a simple, rapid method for detecting various types of Ca2(+)-binding proteins following electrophoresis.     

Intralumenal sarcoplasmic reticulum Ca(2+)-binding proteins

The sarcoplasmic reticulum (SR) controls the level of intracellular Ca2+ in cardiac and skeletal muscle by storing and releasing Ca2+. A set of intralumenal SR Ca(2+)-binding proteins has been identified that may serve important roles in SR Ca2+ storage and mobilization. The most prominent of these SR proteins, calsequestrin, is discretely localized to junctional SR. Other intralumenal proteins are more widely distributed throughout the SR. All of these intralumenal SR Ca(2+)-binding proteins are acidic, stain blue with dye Stains-All, and appear to be substrates for casein kinase II. The biochemistry and cell biology of lumenal SR proteins may conform to a paradigm now emerging from the study of endoplasmic reticulum proteins.     

Supramolecular calsequestrin complex.

As recently demonstrated by overlay assays using calsequestrin-peroxidase conjugates, the major 63 kDa Ca(2+)-binding protein of the sarcoplasmic reticulum forms complexes with itself, and with junctin (26 kDa), triadin (94 kDa) and the ryanodine receptor (560 kDa) [Glover, L., Culligan, K., Cala, S., Mulvey, C. & Ohlendieck, K. (2001) Biochim. Biophys. Acta1515, 120-132]. Here, we show that variations in the relative abundance of these four central elements of excitation-contraction coupling in different fiber types, and during chronic electrostimulation-induced fiber type transitions, are reflected by distinct alterations in the calsequestrin overlay binding patterns. Comparative immunoblotting with antibodies to markers of the junctional sarcoplasmic reticulum, in combination with the calsequestrin overlay binding patterns, confirmed a lower ryanodine receptor expression in slow soleus muscle compared to fast fibers, and revealed a drastic reduction of the RyR1 isoform in chronic low-frequency stimulated tibialis anterior muscle. The fast-to-slow transition process included a distinct reduction in fast calsequestrin and triadin and a concomitant reduction in calsequestrin binding to these sarcoplasmic reticulum elements. The calsequestrin-binding protein junctin was not affected by the muscle transformation process. The increase in calsequestrin and decrease in junctin expression during postnatal development resulted in similar changes in the intensity of binding of the calsequestrin conjugate to these sarcoplasmic reticulum components. Aged skeletal muscle fibers tended towards reduced protein interactions within the calsequestrin complex. This agrees with the physiological concept that the key regulators of Ca(2+) homeostasis exist in a supramolecular membrane assembly and that protein-protein interactions are affected by isoform shifting underlying the finely tuned adaptation of muscle fibers to changed functional demands.     

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.     

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.     

Sequential expression during postnatal development of specific markers of junctional and free sarcoplasmic reticulum in chicken pectoralis muscle.

Skeletal muscle sarcoplasmic reticulum comprises two distinct membrane domains, i.e., the Ca(2+)-pump membrane, corresponding mainly to longitudinal tubules, and the junctional membrane of the terminal cisternae containing the ryanodine receptor/Ca(2+)-release channel. Additional minor proteins previously shown in rabbit fast-twitch skeletal muscle to fractionate selectively to each membrane domain comprise 160- and 53-kDa glycoproteins and 170-kDa low-density lipoprotein (LDL)-binding protein, respectively (Damiani and Margreth, 1991, Biochem. J. 277, 825-832). We report evidence in chicken pectoralis, a predominantly fast muscle, on two closely immunologically related glycoproteins, a minor component of 130-kDa and a major 53-kDa protein. In contrast to the seemingly highly conserved structure of this protein, our results show marked differences in mobilities for chicken 125I-LDL that were detected as a 130- to 116-kDa protein doublet after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, although being otherwise indistinguishable from rabbit 170-kDa protein in LDL-binding characteristics, as well as for preferential association to junctional terminal cisternae. Chicken Ca(2+)-ATPase, although being extensively homologous to rabbit Ca(2+)-ATPase, is shown to be less active and to differ slightly in electrophoretic properties. We have investigated the time course of expression of the specific protein components of longitudinal and of junctional sarcoplasmic reticulum in chick pectoralis muscle from late embryonic development up to 2 months after hatching. Coincident with the posthatching increase in membrane density of high-affinity [3H]ryanodine-binding sites in muscle, both calsequestrin and the species-specific LDL-binding protein(s) are detected in increasing amounts, using ligand blot techniques. In contrast, the appearance and steady accumulation in muscle of Ca(2+)-ATPase, like the time-correlated increase of sarcoplasmic reticulum glycoproteins, are relatively delayed, the most striking changes occurring from 1 week after hatching onward. The sequential expression in chick developing muscle of proteins selectively associated with the junctional terminal cisternae and with longitudinal sarcoplasmic reticulum, respectively, argues for a similar morphogenetic program in avian and mammalian species and, to account for that, for the existence of common epigenetic differentiating influences on the expression of sarcoplasmic reticulum protein genes.     

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.     

Caveolae from canine airway smooth muscle contain the necessary components for a role in Ca(2+) handling

To explain that bronchial smooth muscle undergoes sustained agonist-induced contractions in a Ca(2+)-free medium, we hypothesized that caveolae in the plasma membrane (PM) contain protected Ca(2+). We isolated caveolae from canine tracheal smooth muscle by detergent treatment of PM-derived microsomes. Detergent-resistant membranes were enriched in caveolin-1, a specific marker for caveolae as well as for L-type Ca(2+) channels and Ca(2+) binding proteins (calsequestrin and calreticulin) as determined by Western blotting. Also, the PM Ca(2+) pump was present but not connexin 43 (a noncaveolae PM protein), the sarcoplasmic reticulum (SR) Ca(2+) pump, or the type 1 inositol 1,4, 5-trisphosphate receptor, supporting the idea that SR-derived membranes were not present. Antibodies to caveolin coimmunoprecipitated caveolin with calsequestrin or calreticulin. Thus some of the cellular calsequestrin and calreticulin associated with caveolin on the cytoplasmic face of each caveola. Immunohistochemistry of tracheal smooth muscle crysosections confirmed the localization of caveolin and the PM Ca(2+) pump to the cell periphery, whereas the SR Ca(2+) pump was located deeper in the cell. The presence of L-type Ca(2+) channels, the PM Ca(2+) pump, and the Ca(2+) bindng proteins calsequestrin and calreticulin in caveolin-enriched membranes supports caveola involvement in airway smooth muscle Ca(2+) handling.     

Altered stored calcium release in skeletal myotubes deficient of triadin and junctin

Triadin and junctin are integral sarcoplasmic reticulum membrane proteins that form a macromolecular complex with the skeletal muscle ryanodine receptor (RyR1) but their roles in skeletal muscle calcium homeostasis remain incompletely understood. Here we report that delivery of siRNAs specific for triadin or junctin into C2C12 skeletal myoblasts reduced the expression of triadin and junctin in 8-day-old myotubes by 80 and 100%, respectively. Knocking down either triadin or junctin in these cells reduced Ca2+ release induced by depolarization (10mM KCl) by 20-25%. Unlike triadin knockdown myotubes, junctin knockdown and junctin/triadin double knockdown myotubes also had reduced Ca2+ release induced by 400 microM 4-chloro-m-cresol, 10mM caffeine, 400 microM UTP, or 1 microM thapsigargin. Thus, knocking down junctin compromised the Ca2+ stores in the sarcoplasmic reticulum of these cells. Our subsequent studies showed that in junctin knockdown myotubes at least two sarcoplasmic reticulum proteins (RyR1 and skeletal muscle calsequestrin) were down-regulated while these proteins' mRNA expression was not affected. The results suggest that triadin has a role in facilitating KCl depolarization-induced Ca2+ release in contrast to junctin which has a role in maintaining sarcoplasmic reticulum Ca2+ store size in C2C12 myotubes.     

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum

The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.