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PubMed1993

The endoplasmic-sarcoplasmic reticulum of smooth muscle: immunocytochemistry of vas deferens fibers reveals specialized subcompartments differently equipped for the control of Ca2+ homeostasis.

Cryosection immunofluorescence and immunogold labeling with antibodies against specific markers were used in rat vas deferens smooth muscle fibers to reveal the molecular arrangement of the endomembrane system (referred to variously in the text as ER or sarcoplasmic reticulum [SR]; S-ER or ER/SR) known to participate in the control of Ca2+ homeostasis. The lumenal ER chaperon, immunoglobulin binding protein (BiP), as well as protein disulfide isomerase, and calreticulin, a Ca2+ binding protein expressed by most eukaryotic cells, appeared to be evenly distributed throughout the entire system (i.e., within [a] the nuclear envelope and the few rough-surfaced cisternae clustered near the nucleus; [b] single elements scattered around in the contractile cytoplasm; and [c] numerous, heterogeneous, mainly smooth-surfaced elements concentrated in the peripheral cytoplasm, part of which is in close apposition to the plasmalemma). All other structures, including nuclei, mitochondria, Golgi complex, and surface caveolae were unlabeled. An even distribution throughout the endomembrane system appeared also for the proteins recognized by anti-ER membrane antibodies. In contrast, calsequestrin (the protein that in striated muscles is believed to be the main actor of the rapidly exchanging Ca2+ storage within the lumen of the sarcoplasmic reticulum) was found preferentially clustered at discrete lumenal sites, most often within peripheral smooth-surfaced elements of moderate electron density. Within these elements dual labeling revealed intermixing of calsequestrin with the other lumenal ER proteins. Moreover, the calsequestrin-rich elements were enriched also in the receptor for inositol 1,4,5-trisphosphate, the second messenger that induces Ca2+ release from intracellular stores. These results document the previously hypothesized molecular heterogeneity of the smooth muscle endomembrane system, particularly in relation to the rapid storage and release of Ca2+.

Animals↗
PubMed1998

Reevaluation of the prevalences of serum autoantibodies reactive with "64-kd eye muscle proteins" in patients with thyroid-associated ophthalmopathy.

Serum autoantibodies reactive with eye muscle proteins of "64 kilodaltons (kd)" are frequently found in patients with Graves' hyperthyroidism and thyroid-associated ophthalmopathy (TAO). Earlier, we cloned a 64-kd protein that was identified as calsequestrin, a calcium-binding protein localized in the sarcoplasmic reticulum of striated muscle and extensively studied another cloned 64-kd protein, called 1D, which is expressed in thyroid and eye muscle, and some other tissues. Using a monoclonal antibody against calsequestrin, a polyclonal antibody against 1D and a TAO patient serum reactive with the "64-kd protein," as probes, we performed Western blots of porcine eye muscle membrane. We identified three different proteins in the 63 to 67 kd molecular weight range that were targeted by antibodies in sera from patients with TAO. It was not possible to differentiate antibodies reactive with calsequestrin and 1D because these two proteins have very similar molecular weights--63 to 64 kd--and band appearance in Western blotting. A 67-kd protein was most frequently recognized by TAO patients' sera. Serum antibodies reactive with the 67-kd protein were detected in 73% of patients with active TAO of 1 year duration or less, in 37% of patients with TAO of more than 3 years' duration, in 35% with Graves' hyperthyroidism without evident ophthalmopathy, in 30% of patients with Hashimoto's thyroiditis, and in 16% of normal subjects. Serum antibodies reactive with calsequestrin/1D were detected in 47% of patients with active TAO of less than 1 year, in 22% of patients with TAO longer than 3 years, 17% with Graves' hyperthyroidism without evident ophthalmopathy, in 10% of patients with Hashimoto's thyroiditis, and in 21% of normal subjects. The prevalence of anti-67-kd protein antibodies in TAO patients corresponded to those reactive with the so called "64-kd protein" that we have reported previously. In conclusion, we were able to improve the accuracy of the Western blots by comparing the molecular weight of positive bands using specific antibodies reactive with eye muscle antigens as probes. The previously recognized, and extensively studied, "64-kd protein" is now shown to have a molecular weight of 67 kd. The role of the various eye muscle antibodies in the diagnosis and management of the ophthalmopathy associated with Graves' hyperthyroidism needs to be addressed in prospective studies using purified or recombinant full-length proteins.

Adolescent↗
PubMed2002

Calcium-binding proteins and calcium-release channels in human maturing oocytes, pronuclear zygotes and early preimplantation embryos.

BACKGROUND: The study aim was to investigate the presence and localization of Ca2+-binding proteins and Ca2+-release receptor channels in human maturing oocytes, pronuclear zygotes and preimplantation embryos. METHODS: Immunocytochemical analysis, using specific antibodies against the proteins being studied, followed with confocal laser microscopy, was performed on human oocytes and embryos. RESULTS: Calreticulin and calsequestrin (the two major calcium storage proteins of somatic cells), two types of calcium release receptors, the inositol trisphosphate and ryanodine receptors (InsP(3)R-2, RyRs-1,2,3), and the molecular chaperone, calnexin, were identified in all investigated cell types. Calreticulin was predominant in the cell cortex and in the nuclear envelope, while calsequestrin was distributed throughout the entire cytoplasm. Generally, localization of the InsP(3)R-2 and RyRs was similar to that of calreticulin and calsequestrin respectively. Both types of receptor were enriched in the subplasmalemmal region of meiotic oocytes. In addition, the InsP(3)R was detected in the nuclear structures of oocytes and blastomeres. Calnexin distribution overlapped with that of calreticulin but appeared to be present in distinct subcompartments. CONCLUSIONS: Human oocytes and embryos express the calcium sequestration and release proteins in highly organized and developmentally regulated patterns. Fine-tuning of these proteins may play a crucial role in regulation of Ca2+ transience during oocyte maturation, fertilization and early embryo development.

Blastocyst↗
PubMed1997

Alterations in expression of sarcoplasmic reticulum gene in Dahl rats during the transition from compensatory myocardial hypertrophy to heart failure.

OBJECTIVES: To clarify whether the functional changes during the transition from compensatory myocardial hypertrophy to failure are associated with changes in sarcoplasmic reticulum gene expression. METHODS: We examined the gene expression of sarcoplasmic reticulum proteins [sarcoplasmic reticulum Ca2+-ATPase (SERCA), phospholamban, calsequestrin and ryanodine receptor] in Dahl salt-sensitive (Dahl-S) rats fed a high-salt (8%) diet from the age of 6 weeks. In-vivo contractile functioning was evaluated using echocardiography, and gene expression of sarcoplasmic reticulum proteins in the left ventricle was analyzed by Northern blotting for each stage of left ventricular hypertrophy. RESULTS: SERCA messenger RNA (mRNA) levels in Dahl-S rats with compensatory hypertrophy did not change significantly, whereas phospholamban mRNA levels were increased by 61% (P < 0.01), and calsequestrin mRNA levels were increased by 130% (P < 0.01) compared with those in Dahl salt-resistant (Dahl-R) rats. SERCA mRNA levels in Dahl-S rats with decompensated dilatation were decreased by 32% (P< 0.05), whereas levels of phospholamban and calsequestrin mRNA remained unchanged. Ryanodine receptor mRNA levels did not change either with compensatory hypertrophy or with decompensated dilatation. CONCLUSIONS: Alterations in expression of sarcoplasmic reticulum gene may be related to changes in systolic and diastolic properties in compensatory hypertrophy and heart failure.

Animals↗
PubMedDate not supplied

Calcium storage in nonmuscle tissues: is the retina special?

It is now well established that calreticulin is a high capacity Ca(2+)-binding protein which is a major Ca2+ storage protein of the lumen of endoplasmic reticulum membranes in a wide variety of tissues with the exception of skeletal and cardiac muscles. However, in nervous tissue, confusion exists regarding the nature of the intracellular Ca2+ stores, as the organelle responsible for Ca2+ storage has been identified as the endoplasmic reticulum by some investigators and as the specialized organelle, calciosome by others. Calreticulin, calsequestrin, and calsequestrin-like proteins have all been, on different occasions, reported to be present in calciosomes. Cerebral and cerebellar tissues, moreover, have been shown to contain somewhat different systems of Ca(2+)-buffering proteins. In the present paper we discuss evidence that the Ca2+ storage systems of the retina may prove to be more complex than those of other neuronal tissues. Biochemical and immunocytochemical evidence indicates the presence of either an isoform of calreticulin or another protein that is antigenically similar to calreticulin, but of slightly higher molecular weight, in the endoplasmic reticulum of both neurons and Müller glia from rabbit neural retina. However, as retinal neurons express Purkinje cell markers, one may expect to observe the presence of calsequestrin in these cells as well. Secondly, antibodies against the onchocercal RAL-1 antigen recognize a protein sharing 62-65% amino acid sequence identity with calreticulin. The anti-RAL-1 antibodies show specificity for the retina. Whether or not the RAL-1 antigen is an active part of the Ca2+ storage systems of the retina remains to be verified.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1997

Calreticulin, a potential vascular regulatory protein, reduces intimal hyperplasia after arterial injury.

Both thrombotic and inflammatory responses to arterial injury have been implicated in atherosclerotic plaque growth. Calreticulin is a ubiquitous calcium-binding protein with antithrombotic activity and, in addition, is associated with leukocyte activation. We are investigating calreticulin as a potential vascular regulatory protein. The development of intimal hyperplasia was studied at sites of balloon injury in iliofemoral arteries from 91 rats. Calreticulin was infused directly into the artery immediately before balloon injury, and plaque growth was then assessed at 4 weeks' follow-up. Parallel studies of the effects of each calreticulin domain as well as a related calcium-binding protein, calsequestrin, were examined. The effects of calreticulin on platelet activation, clot formation, and mononuclear cell migration were also studied. When infused before balloon injury in rat iliofemoral arteries, calreticulin, or its high-capacity Ca(2+)-binding C domain, significantly reduces plaque development, whereas calsequestrin, a related calcium-binding protein that lacks the multifunctional nature of calreticulin, does not decrease plaque area (saline: 0.037 +/- 0.007 mm2, calsequestrin: 0.042 +/- 0.021 mm2, calreticulin: 0.003 +/- 0.002 mm2, n = 46, P < .04). The N domain and more specifically the P domain, a low-capacity, high-affinity calcium-binding domain in calreticulin, do not reduce intimal hyperplasia (N + P domain: 0.038 +/- 0.012 mm2, C domain: 0.003 +/- 0.002 mm2, n = 45 rats, P < .0001). Calreticulin reduces macrophage and T cell staining in the arterial wall after injury but has no direct effect on monocyte migration in vitro (percent medial area staining positive for macrophage 24 hours after injury (N + P: 4.06 +/- 1.42, calreticulin: 0.273 +/- 0.02; n = 26, P < .009). Calreticulin does, however, reduce platelet-dependent whole blood clotting time, in vitro (baseline: 78.23 +/- 2.04 seconds, calreticulin: 113.5 +/- 1.95 seconds; n = 5, P < .002). We conclude that calreticulin significantly reduces intimal hyperplasia after arterial injury, potentially acting as a vascular regulatory protein.

Angioplasty, Balloon↗
PubMed2004

Organization of Ca2+ release units in excitable smooth muscle of the guinea-pig urinary bladder.

Ca(2+) release from internal stores (sarcoplasmic reticulum or SR) in smooth muscles is initiated either via pharmaco-mechanical coupling due to the action of an agonist and involving IP3 receptors, or via excitation-contraction coupling, mostly involving L-type calcium channels in the plasmalemma (DHPRs), and ryanodine receptors (RyRs), or Ca(2+) release channels of the SR. This work focuses attention on the structural basis for the coupling between DHPRs and RyRs in phasic smooth muscle cells of the guinea-pig urinary bladder. Immunolabeling shows that two proteins of the SR: calsequestrin and the RyR, and one protein the plasmalemma, the L-type channel or DHPR, are colocalized with each other within numerous, peripherally located sites located within the caveolar domains. Electron microscopy images from thin sections and freeze-fracture replicas identify feet in small peripherally located SR vesicles containing calsequestrin and distinctive large particles clustered within small membrane areas. Both feet and particle clusters are located within caveolar domains. Correspondence between the location of feet and particle clusters and of RyR- and DHPR-positive foci allows the conclusion that calsequestrin, RyRs, and L-type Ca(2+) channels are associated with peripheral couplings, or Ca(2+) release units, constituting the key machinery involved in excitation-contraction coupling. Structural analogies between smooth and cardiac muscle excitation-contraction coupling complexes suggest a common basic mechanism of action.

Animals↗
PubMedDate not supplied

Sarcoplasmic reticulum membrane and heart development.

Intracellular Ca2+ concentrations in cardiac cells are dependent on trans-sarcolemmal Ca2+ fluxes and the ability of sarcoplasmic reticulum to release and take up Ca2+. Ca2+ accumulation by sarcoplasmic reticulum membranes causes muscle to relax, whereas Ca2+ release from sarcoplasmic reticulum initiates contraction. Ca2+ transport by the sarcoplasmic is mediated by a Ca2+-dependent ATPase enzyme. Ca2+ release from sarcoplasmic reticulum may be mediated by a ligant-gated Ca2+ channel. The physiological role of sarcoplasmic reticulum in developing muscle is not well established. In this report we investigated the composition and function of sarcoplasmic reticulum membranes during cardiac myogenesis. Phospholamban, a major phosphoprotein in mature sarcoplasmic reticulum membranes was present during early stages of cardiac myogenesis. The embryonic form of phospholamban was phosphorylated by cAMP-dependent protein kinase but not in the presence of Ca2+ and calmodulin. Ca2+ uptake and Ca2+-dependent ATPase activity were low in fetal sarcoplasmic reticulum compared to adult control membranes, although the apparent affinities of the enzyme for Ca2+ were similar. Sarcoplasmic reticulum vesicles used in these studies had very low levels of plasma membrane and mitochondrial contamination. The amounts of both 110-kDa Ca2+-ATPase and 55-kDa calsequestrin in the sarcoplasmic reticulum membrane were lower in fetal sarcoplasmic reticulum vesicles compared to mature membranes. Ca2+-ATPase and calsequestrin were identified in the isolated sarcoplasmic reticulum vesicles using specific antibodies produced against these membrane proteins. Age-related differences in Ca2+ transport properties of cardiac sarcoplasmic reticulum and in the amount of Ca2+-ATPase and calsequestrin may explain alterations in the regulation of intracellular Ca2+ concentrations in fetal heart muscle. This may relate to the developmental changes observed in myocardial function.

Adenosine Triphosphatases↗
PubMed1980

Assembly of the sarcoplasmic reticulum. Biosynthesis of the high affinity calcium binding protein in rat skeletal muscle cell cultures.

Temporal patterns of biosynthesis of the high affinity calcium binding protein from the sarcoplasmic reticulum were determined and compared with rates of ATPase ane cells. Cells at various stages of differentiation were incubated for 2 h with [35S]methionine. Specific proteins were isolated from detergent extracts of cells by incubation with antibodies specific against the various proteins and immunoprecipitates were separated by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Radioactivity incorporated into specific bands was analyzed by counting gel slices and incorporation data were used to obtain relative rates of individual protein sys found to be indistinguishable from that of calsequestrin when cells were grown in standard medium, in medium containing 60 microM Ca2+ which prevented fusion of cells, or in enriched medium which delayed cell fusion. The high affinity calcium binding protein had a relatively high turnover rate with a half-life of about 10 h. These studies suggest that synthesis of calsequestrin and the high affinity calcium binding protein are coordinated even though calsequestrin is a glycoprotein, whereas the high affinity calcium binding protein is not glycosylated.

Adenosine Triphosphatases↗
PubMed1998

Eye muscle antibodies in patients with ocular myasthenia gravis: possible mechanism for eye muscle inflammation in acetylcholine-receptor antibody-negative patients.

Myasthenia gravis is an organ-specific autoimmune disorder generally thought to be caused by an antibody-mediated attack against the skeletal muscle nicotinic acetylcholine (Ach) receptor (AchR) at the neuromuscular junction. Extraocular muscle weakness and double vision are present in about 90% of patients with myasthenia gravis and are the predominant complaints in about 20% of patients, when the condition is called ocular myasthenia gravis (OMG). While serum antibodies against the AchR are detected in most patients with generalized myasthenia gravis (GMG), they are not found in about one-third of patients with the ocular variety, and epidemiological, clinical, and serological studies suggest that OMG and GMG are two separate diseases. Both forms of myasthenia gravis are sometimes associated with thyroid autoimmunity or thyroid-associated ophthalmopathy (TAO). We have therefore tested the sera of patients with GMG and OMG by Western blotting for antibodies against porcine eye muscle membrane proteins in general, and by enzyme-linked immunosorbent assays (ELISA) specifically for reaction with two skeletal muscle antigens which are prominent marker antigens for TAO, namely, the calcium-binding protein calsequestrin and the so-called "64-kDa protein." The 64-kDa protein has recently been identified as the flavoprotein subunit of mitochondrial succinate dehydrogenase. Patients with ophthalmopathy and myasthenia were excluded. Nine of the patients had associated Graves' hyperthyroidism without evident ophthalmopathy and one had Hashimoto's thyroiditis. Antibodies against porcine eye muscle membrane antigens of M(r) 15-110 kDa were detected in patients with GMG or OMG, one or more antibodies being detected in 100% of patients with GMG and in 88% of those with OMG. The most frequently found antibodies were those targeting eye muscle membrane proteins of 15, 67, and 110 kDa. Antibodies reactive with purified calsequestrin (63 kDa) were detected in 21% of patients with OMG but in no patient with GMG. Antibodies recognizing purified succinate dehydrogenase (67 kDa) were found in 42% of patients with OMG, in 100% (5 of 5) of patients with GMG, and in 48% of all patients with myasthenia gravis not associated with Graves' hyperthyroidism. There was no close correlation between any eye muscle-reactive antibody and antibodies against the AchR in either group of myasthenic patients. The findings support the notion that immunoreactivity against skeletal muscle proteins other than the AchR may play a role in the development of the muscle weakness in AchR antibody-negative patients with OMG and GMG, although it is unlikely that any of the antibodies demonstrated in this study are directly implicated. Similarly, while the demonstration of antibodies reactive with eye muscle antigens associated with TAO in patients with OMG raises the possibility that the link between the ocular lesions of myasthenia gravis and Graves' disease may be autoimmunity against a common antigen(s), it is more likely that both disorders are mediated by cytotoxic T cells recognizing another cell membrane antigen, such as the novel thyroid and eye muscle shared protein G2s, and that serum antibodies reactive with succinate dehydrogenase Fp subunit and calsequestrin are markers of an immune-mediated eye muscle reaction.

Animals↗
PubMed1988

Calcium binding protein changes of sarcoplasmic reticulum from rat denervated skeletal muscle.

Two Ca2+ sequestering proteins were studied in fast-twitch (EDL) and slow-twitch (soleus) muscle sarcoplasmic reticulum (SR) as a function of denervation time. Ca2+-ATPase activity measured in SR fractions of normal soleus represented 5% of that measure in SR fractions of normal EDL. Denervation caused a severe decrease in activity only in fast-twitch muscle. Ca2+-ATPase and calsequestrin contents were affected differently by denervation. In EDL SR, Ca2+-ATPase content decreased progressively, whereas in soleus SR, no variation was observed. Calsequestrin showed a slight increase in both muscles as a function of denervation time correlated with increased 45Ca-binding. These results indicate first that Ca2+-ATPase activity in EDL was under neural control, and that because of low Ca2+-ATPase activity and content in slow-twitch muscle no variation could be detected, and secondly that greater calsequestrin content might represent a relative increasing of heavy vesicles or decreasing of light vesicles as a function of denervation time in the whole SR fraction isolated in both types of muscles.

Animals↗
PubMed2004

Eye muscle antibodies in Graves' ophthalmopathy: pathogenic or secondary epiphenomenon?

The extra ocular (eye) muscles are one of the principal tissues involved in the autoimmune-mediated inflammation of Graves' ophthalmopathy (GO). Several eye muscle proteins are targeted by autoantibodies or sensitized T lymphocytes, or both, and include: G2s, which is now identified as the terminal 141 amino acids of the winged-helix transcription factor FOXP1, the flavoprotein (Fp) subunit of the mitochondrial enzyme succinate dehydrogenase, the so-called "64kDa protein", a non-tissue specific membrane protein called 1D and the calcium binding protein calsequestrin. Of these, antibodies against G2s and Fp are the most sensitive markers of eye muscle damage in patients with thyroid autoimmunity even though neither antigen is specific to eye muscle and neither antibody is specific to GO. However, the recent finding that the calsequestrin gene is 4.7 times more expressed in eye muscles than other skeletal muscles suggests that we should reconsider the possible role of anti-calsequestrin autoantibodies in ophthalmopathy. GO may comprise two main subtypes with different pathogenetic mechanisms, namely ocular myopathy in which eye muscle inflammation predominates and congestive ophthalmopathy where inflammatory changes occur in the periorbital connective tissues in the absence of eye muscle dysfunction. Anti-G2s and anti-Fp antibodies are closely associated with the ocular myopathy subtype of GO while antibodies targeting type XIII collagen, the only member of the collagen family to have a transmembrane domain, are closely linked to congestive ophthalmopathy. Since both G2s and Fp are intracellular antigens it is unlikely that either antibody causes eye muscle fiber damage in GO, although a role in the later stages of the disease when the fiber has released its cellular contents has not been excluded. Eye muscle antibodies that are cytotoxic to eye muscle cells in antibody-dependent cell-mediated cytotoxicity (ADCC) are more likely to play a role in eye muscle fiber damage since they target a putative eye muscle cell membrane antigen, the identity of which is currently being investigated. While anti-G2s and anti-Fp antibodies are probably secondary to an underlying reaction, such as cytotoxic T lymphocyte targeting of an eye muscle membrane antigen that has yet to be identified, they are reliable markers of immunologically mediated eye muscle fiber damage in patients with Graves' hyperthyroidism. In conclusion, while a pathogenic role for eye muscle antibodies has not been excluded, they are most likely secondary to cytotoxic T cell reactions in GO and, as such, good markers of this autoimmune disease.

Animals↗
PubMed1991

Identification and immunolocalization of calreticulin in pancreatic cells: no evidence for "calciosomes".

In the present study, we have shown that calreticulin is a major Ca(2+)-sequestering protein in pancreatic microsomes. This protein is a peripheral membrane protein and could be extracted from the microsomal membrane with carbonate buffer at pH 11.4. Calreticulin was identified in the membrane fractions by immunoblotting with a specific antibody, by a 45Ca2+ overlay technique, and by NH2-terminal amino acid analysis of the purified protein. Immunocytochemical localization of calreticulin in pancreatic acinar cells and pancreatic fibroblasts showed that the protein is localized to the ER membranes in these cells. We were unable to detect calsequestrin or any calsequestrin-like proteins in the pancreas and found no evidence for the existence of large numbers of specialized, calreticulin-containing vesicles which could be an equivalent of the calsequestrin-containing calciosomes previously reported in this tissue. Purified pancreatic calreticulin binds Ca2+ with both a low and a high capacity (approximately 1 mol of Ca2+/mol of protein and approximately 20-23 mol of Ca2+/mol of protein). The concentrations of Ca2+ required for half-maximal saturation of the low and high capacity sites were approximately 4-6 microM and approximately 1.5 mM, respectively. We conclude that calreticulin, which is confined to the lumen of the ER, plays a major role in Ca2+ storage in pancreatic cells.

Amino Acid Sequence↗
PubMed2004

A proton-led model of fast calcium waves.

Fast (10-30 microm/s) calcium waves can be propagated through all nucleated eukaryotic cells that have been tested as well as certain cell-free extracts. In a widely used model, they are propagated by a reaction-diffusion cycle in which calcium ions diffuse along the outside of endoplasmic reticula and induce their own release from calsequestrin or calreticulin molecules stored within the reticulum's lumen. Here we propose a new tandem wave model in which they are also propagated by a reaction-diffusion cycle within a reticulum's lumen. In this cycle, increases in luminal [H(+)] induce proton release from luminal calsequestrin or calreticulin. The released protons diffuse ahead to where they release more protons from these luminal storage proteins. What might be called proton induced proton release. They also raise luminal electropositivity. The resultant luminal waves are coordinated with extrareticular ones by movements of calcium and hydrogen ions through the reticular membrane. This model makes five testable predictions which include the autorelease of protons in solutions of calsequestrins or calreticulins as well as waves of increased [H(+)], of increased [Ca(2+)] and of more positive voltage within the reticula of whole cells. Moreover, under some conditions, such luminal waves should cross regions without cytosolic ones.

Animals↗
PubMed1999

Self-aggregation of triadin in the sarcoplasmic reticulum of rabbit skeletal muscle.

The 95 kDa transmembrane glycoprotein triadin is believed to be an essential component of excitation-contraction coupling in the junctional sarcoplasmic reticulum of skeletal muscle fibers. It is debatable whether triadin mediates intraluminal interactions between calsequestrin and the ryanodine receptor exclusively or whether this junctional protein provides also a cytoplasmic linkage between the Ca2+-release channel and the dihydropyridine receptor. Here, we could show that native triadin exists as disulfide-linked homo-polymers of above 3000 kDa. Under non-reducing conditions, protein bands representing the alpha1-dihydropyridine receptor and calsequestrin did not show an immunodecorative overlap with the extremely high-molecular-mass triadin clusters. Following chemical crosslinking, the ryanodine receptor and triadin exhibited a similarly decreased electrophoretic mobility. However, immunoblotting of diagonal non-reducing/reducing two-dimensional gels clearly demonstrated a lack of overlap between the immunodecorated bands representing triadin, the alpha1-dihydropyridine receptor, the ryanodine receptor and calsequestrin. Thus, in native membranes triadin appears to form large self-aggregates primarily. Although triadin exists in a close neighborhood relationship to the Ca2+-release channel tetramers, it does not seem to be directly linked to the other main triad components implicated in the regulation of the excitation-contraction-relaxation cycle and Ca2+-homeostasis. This agrees with a proposed role of triadin in the maintenance of overall triad architecture.

Animals↗
PubMed2006

Depletion "skraps" and dynamic buffering inside the cellular calcium store.

Ca2+ signals, produced by Ca2+ release from cellular stores, switch metabolic responses inside cells. In muscle, Ca2+ sparks locally exhibit the rapid start and termination of the cell-wide signal. By imaging Ca2+ inside the store using shifted excitation and emission ratioing of fluorescence, a surprising observation was made: Depletion during sparks or voltage-induced cell-wide release occurs too late, continuing to progress even after the Ca2+ release channels have closed. This finding indicates that Ca2+ is released from a "proximate" compartment functionally in between store lumen and cytosol. The presence of a proximate compartment also explains a paradoxical surge in intrastore Ca2+, which was recorded upon stimulation of prolonged, cell-wide Ca2+ release. An intrastore surge upon induction of Ca2+ release was first reported in subcellular store fractions, where its source was traced to the store buffer, calsequestrin. The present results update the evolving concept, largely due to N. Ikemoto and C. Kang, of calsequestrin as a dynamic store. Given the strategic location and reduction of dimensionality of Ca2+-adsorbing linear polymers of calsequestrin, they could deliver Ca2+ to the open release channels more efficiently than the luminal store solution, thus constituting the proximate compartment. When store depletion becomes widespread, the polymers would collapse to increase store [Ca2+] and sustain the concentration gradient that drives release flux.

Animals↗
PubMed1998

Role of the eye muscles in thyroid eye disease: identification of the principal autoantigens.

Thyroid-associated ophthalmopathy (TAO) is a progressive orbital disorder associated with Graves' hyperthyroidism and, less often, Hashimoto's thyroiditis in which autoantibodies react with orbital antigens and lead to exophthalmos and eye muscle inflammation. Eye muscle (EM) membrane proteins initially reported as 55 and 64 kd are the best markers of ophthalmopathy. The "64-kd protein" is now shown to be the flavoprotein subunit of mitochondrial succinate dehydrogenase and to have a correct molecular weight of 67 kd. We have cloned a fragment of a novel eye muscle protein, which we call G2s, and sequenced 1.4 kb of the full length cDNA. G2s does not share any significant homologies with other reported proteins. The 5.9 kb G2s mRNA, that corresponds to a protein of approximately 220 kd, is expressed in EM, other skeletal muscle and thyroid, but not in other tissues tested. We have also cloned and sequenced a 63-kd eye muscle protein identified as the calcium binding protein calsequestrin. Antibodies against calsequestrin were found in 40% of patients with active ophthalmopathy, but in 0% of normal subjects. Finally, we have sequenced a 19 amino acid fragment of a 55-kd porcine eye muscle membrane protein that exactly matched porcine and human sarcalumenin, a 160-kd glycoprotein localized in the lumen of the longitudinal sarcoplasmic reticulum of the skeletal muscle fiber where it binds calcium. A 53-kd glycoprotein fragment of the molecule corresponds to the 55-kd protein. In a preliminary study, serum antibodies against purified sarcalumenin were detected in 40% of patients with active TAO of less than 1 year duration, but in no controls tested. We porpose that the primary autoantigen in TAO is G2s, which would also explain the association of ophthalmopathy with thyroid autoimmunity, and that antibodies against the intracellular proteins flavoprotein, calsequestrin, and sarcalumenin are secondary markers of an immune-mediated reaction in eye muscle in patients with thyroid autoimmunity.

Autoantigens↗
PubMed1997

Alterations in sarcoplasmic reticulum calcium-storing proteins in pressure-overload cardiac hypertrophy.

The alterations of intracellular calcium (Ca2+) homeostasis may be responsible for the contractile defects in pressure-overload cardiac hypertrophy. The Ca(2+)-adenosinetriphosphatase (ATPase) protein level of the sarcoplasmic reticulum (SR) is reduced in the hypertrophied or failing heart. However, it is not known whether Ca(2+)-storing proteins, including calsequestrin and calreticulin, are also altered during cardiac hypertrophy. We quantified SR Ca(2+)-regulatory proteins using Western blot analysis in left ventricular (LV) muscle isolated from sham-operated control rats (n = 6) and rats with pressure overload 4 wk after abdominal aortic constriction (n = 7). The contractile function of isolated LV myocytes, assessed by the sarcomere motion measured with laser diffraction, was depressed in aortic-constricted rats. The SR Ca(2+)-ATPase protein level was decreased to 56 +/- 9% (SE) of the control value in hypertrophied myocardium (P < 0.01). The calsequestrin protein level was not altered, whereas calreticulin was increased by 120 +/- 3% of the control value in aortic-constricted rats (P < 0.05). The alterations in SR Ca(2+)-regulatory proteins were equally observed in hypertrophied hearts even when the results were normalized using the amounts of myosin heavy chain proteins in each sample. Immunohistochemical staining of calsequestrin in the control heart showed cross striations at the Z lines, whereas calreticulin was hardly observed within myocytes but was intense within interstitial fibroblasts. In the hypertrophied heart, calreticulin was observed at the perinuclear region within the myocyte cytoplasm. These data indicate that pressure-overload cardiac hypertrophy causes the alterations in SR Ca(2+)-storing proteins as well as in Ca(2+)-ATPase, which may contribute to the contractile dysfunction of the hypertrophied myocytes.

Animals↗