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PubMed2004

Comparing skeletal and cardiac calsequestrin structures and their calcium binding: a proposed mechanism for coupled calcium binding and protein polymerization.

Calsequestrin, the major calcium storage protein of both cardiac and skeletal muscle, binds and releases large numbers of Ca(2+) ions for each contraction and relaxation cycle. Here we show that two crystal structures for skeletal and cardiac calsequestrin are nearly superimposable not only for their subunits but also their front-to-front-type dimers. Ca(2+) binding curves were measured using atomic absorption spectroscopy. This method enables highly accurate measurements even for Ca(2+) bound to polymerized protein. The binding curves for both skeletal and cardiac calsequestrin were complex, with binding increases that correlated with protein dimerization, tetramerization, and oligomerization. The Ca(2+) binding capacities of skeletal and cardiac calsequestrin are directly compared for the first time, with approximately 80 Ca(2+) ions bound per skeletal calsequestrin and approximately 60 Ca(2+) ions per cardiac calsequestrin, as compared with net charges for these molecules of -80 and -69, respectively. Deleting the negatively charged and disordered C-terminal 27 amino acids of cardiac calsequestrin results in a 50% reduction of its calcium binding capacity and a loss of Ca(2+)-dependent tetramer formation. Based on the crystal structures of rabbit skeletal muscle calsequestrin and canine cardiac calsequestrin, Ca(2+) binding capacity data, and previous light-scattering data, a mechanism of Ca(2+) binding coupled with polymerization is proposed.

Amino Acid Sequence↗
PubMed2005

Regulation of ryanodine receptors by calsequestrin: effect of high luminal Ca2+ and phosphorylation.

Calsequestrin, the major calcium sequestering protein in the sarcoplasmic reticulum of muscle, forms a quaternary complex with the ryanodine receptor calcium release channel and the intrinsic membrane proteins triadin and junctin. We have investigated the possibility that calsequestrin is a luminal calcium concentration sensor for the ryanodine receptor. We measured the luminal calcium concentration at which calsequestrin dissociates from the ryanodine receptor and the effect of calsequestrin on the response of the ryanodine receptor to changes in luminal calcium. We provide electrophysiological and biochemical evidence that: 1), luminal calcium concentration of >/=4 mM dissociates calsequestrin from junctional face membrane, whereas in the range of 1-3 mM calsequestrin remains attached; 2), the association with calsequestrin inhibits ryanodine receptor activity, but amplifies its response to changes in luminal calcium concentration; and 3), under physiological calcium conditions (1 mM), phosphorylation of calsequestrin does not alter its ability to inhibit native ryanodine receptor activity when the anchoring proteins triadin and junctin are present. These data suggest that the quaternary complex is intact in vivo, and provides further evidence that calsequestrin is involved in the sarcoplasmic reticulum calcium signaling pathway and has a role as a luminal calcium sensor for the ryanodine receptor.

Acid Phosphatase↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1994

Organization of calsequestrin-positive sarcoplasmic reticulum in rat cardiomyocytes in culture.

The sarcoplasmic reticulum (SR) regulates the levels of cytoplasmic free Ca2+ ions in muscle cells. Calsequestrin is a major Ca(2+)-storing protein and is localized at special sites in the SR. To investigate the development of calsequestrin-positive SR and its interaction with the cytoskeleton, we examined the distribution of calsequestrin in cultured cardiomyocytes from newborn rats by immunofluorescence with anticalsequestrin and antitubulin antibodies and rhodamine-phalloidin. In frozen sections of neonatal rat heart, anticalsequestrin immunostaining was apparent as cross-striations at Z-lines. When newborn cardiomyocytes were isolated, calsequestrin-positive SR was disorganized and was apparent as small vesicles beneath the sarcolemma, whereas myofibrils accumulated in the center of the cells. As the cells spread in culture, calsequestrin-positive vesicles spread to the periphery of the cytoplasm, becoming associated with the developing myofibrils. In mature cells, calsequestrin was closely associated with myofibrils, showing cross-striations at the Z-lines. Double-labeling using anticalsequestrin and antitubulin antibodies demonstrated that the distribution of calsequestrin-positive structures was similar to that of the microtubular arrays. When the microtubules were depolymerized by nocodazole at an early stage, the extension of the SR to the cell periphery was inhibited. In mature cardiomyocytes, nocodazole appeared not to affect the distribution of the SR. These results indicate that the calsequestrin-positive SR in cardiomyocytes is organized at the proper sites of myofibrils during myofibrillogenesis and that the microtubules might serve as tracts for the transport of components of the SR.

Animals↗
PubMed2000

Cardiac-specific overexpression of calsequestrin results in left ventricular hypertrophy, depressed force-frequency relation and pulsus alternans in vivo.

Cardiac-specific overexpression of calsequestrin has been shown to result in significant decreases in contractile parameters and intracellular Ca(2+)transients in vitro. Therefore, the purpose of the present study was to determine the effects of calsequestrin overexpression on basal cardiac function and the force-frequency relation in vivo. Calsequestrin overexpression mice (CSQ-OE, n=20) and their isogenic controls (WT) were studied with an integrative approach using transthoracic echocardiography, stress-shortening relations, and invasive hemodynamics in intact closed-chest mice. M-mode echocardiography indicated that calsequestrin overexpression resulted in concentric hypertrophy (+52%) and an increase in LV ejection phase indices. However, mean end-systolic stress-shortening coordinates revealed that at matched end-systolic wall-stress, fractional shortening was depressed in CSQ-OE mice. This was confirmed by depressed indices of LV isovolumic contraction and relaxation in CSQ-OE v. WT mice. Furthermore, overexpression of calsequestrin resulted in a downward and leftward shift of the biphasic force-frequency relation; thus, the critical heart (HR(crit)) was significantly lower in calsequestrin-overexpression mice (264+/-15 bpm) than in wild-type controls (365+/-21 bpm). Surprisingly, calsequestrin overexpression was associated with the induction of pulsus alternans in every animal (at an average heart rate of 428+/-26 bpm), whereas none of the wild-type controls displayed this phenomenon. We conclude that: (i) although increased levels of calsequestrin result in decreased myocardial contractility and a depressed force-frequency relation, LV wall stress is reduced and chamber function is normal, and (ii) an increase in SR Ca(2+)storage capacity induces pulsus alternans in the intact anesthetized mouse.

Animals↗
PubMed1991

Purification and characterization of a calsequestrin-like calcium-binding protein from carp (Cyprinus carpio) sarcoplasmic reticulum.

1. A calsequestrin-like calcium-binding protein was purified from carp sarcoplasmic reticulum by column chromatographies using DEAE-cellulose and Butyl-Toyopearl 650S. 2. The mol. wt was estimated to be 50 kDa, which was larger than that of rabbit calsequestrin (42 kDa). 3. Carp calsequestrin-like protein bound Ca2+ with a higher affinity (apparent Kd = 400 microM) and lower capacity (25 mol/mol) compared with rabbit calsequestrin (1 mM and 40-50 mol/mol, respectively). 4. Anti-carp calsequestrin-like protein rabbit antiserum reacted with rabbit calsequestrin in immunoblotting analysis. 5. Carp calsequestrin-like protein was rich in acidic amino acids, as was rabbit calsequestrin.

Amino Acids↗
PubMed2003

Effects of prenatal glucocorticoid exposure on cardiac calreticulin and calsequestrin protein expression during early development and in adulthood.

Overexpression of the conserved Ca(2+)-binding proteins calreticulin and calsequestrin impairs cardiac function, leading to premature death. Calreticulin is vital for embryonic development, but also impairs glucocorticoid action. Glucocorticoid overexposure during late fetal life causes intra-uterine growth retardation and programmed hypertension in adulthood. To determine whether intra-uterine growth retardation or programmed hypertension was associated with altered calreticulin or calsequestrin expression, effects of prenatal glucocorticoid overexposure (maternal dexamethasone treatment on days 15-21 of pregnancy) were examined during fetal life and postnatal development until adulthood (24 weeks). Dexamethasone (100 or 200 microg/kg of maternal body weight) was administered via osmotic pump. Calreticulin was detected as a 55 kDa band and calsequestrin as 55 and 63 kDa bands in 21 day fetal hearts. Only the 55 kDa calsequestrin band was detected postnatally. Prenatal glucocorticoid overexposure at the higher dose decreased calreticulin protein expression (26%; P <0.05) but increased calsequestrin protein expression, both 55 and 63 kDa bands, by 87% ( P <0.01) and 78% ( P <0.01); only the 55 kDa calsequestrin band was increased at the lower dose (66%; P <0.05). Offspring of dams treated at the lower dexamethasone dose were studied further. In control offspring, cardiac calreticulin protein expression declined between 2 and 3 weeks of age, and remained suppressed until adulthood. Cardiac calsequestrin protein expression increased 2-fold between fetal day 21 and postnatal day 1 and continued to increase until adulthood, at which time it was 3.4-fold higher ( P <0.001). Prenatal dexamethasone exposure minimally affected postnatal calsequestrin protein expression, but the postnatal decline in calreticulin protein expression was abrogated and calreticulin protein expression in adulthood was 2.2-fold increased ( P <0.001) compared with adult controls. In view of the known associations between cardiac calreticulin overexpression and impaired cardiac function, targeted up-regulation of calreticulin may contribute to the increased risk of adult heart disease introduced as a result of prenatal overexposure to glucocorticoids.

Animals↗
PubMed2002

Mass spectrometry of cardiac calsequestrin characterizes microheterogeneity unique to heart and indicative of complex intracellular transit.

Cardiac calsequestrin concentrates in junctional sarcoplasmic reticulum in heart and skeletal muscle cells by an undefined mechanism. During transit through the secretory pathway, it undergoes an as yet uncharacterized glycosylation and acquires phosphate on CK2-sensitive sites. In this study, we have shown that active calsequestrin phosphorylation occurred in nonmuscle cells as well as muscle cells, reflecting a widespread cellular process. To characterize this post-translational modification and resolve individual molecular mass species, we subjected purified calsequestrin to mass spectrometry using electrospray ionization. Mass spectra showed that calsequestrin glycan structure in nonmuscle cells was that expected for an endoplasmic reticulum-localized glycoprotein and showed that each glycoform existed as four mass peaks representing molecules that also had 0-3 phosphorylation sites occupied. In heart, mass peaks indicated carbohydrate modifications characteristic of transit through Golgi compartments. Phosphorylation did not occur on every glycoform present, suggesting a far more complex movement of calsequestrin molecules in heart cells. Significant amounts of calsequestrin contained glycan with only a single mannose residue, indicative of a novel post-endoplasmic reticulum mannosidase activity. In conclusion, glyco- and phosphoforms of calsequestrin chart a complex cellular transport in heart, with calsequestrin following trafficking pathways not present or not accessible to the same molecules in nonmuscle.

Animals↗
PubMed1976

Assembly of the sarcoplasmic reticulum. Biosynthesis of calsequestrin in rat skeletal muscle cell cultures.

Temporal patterns of biosynthesis of the sarcoplasmic reticulum protein, calsequestrin, were analyzed and compared with rates of ATPase synthesis in primary cultures of rat skeletal muscle cells. Rates of synthesis were measured by the incorporation of radioactive leucine into the isolated proteins. Cells at various stages of differentiation were incubated for 2 h with tritium-labeled leucine and extracted with detergent. The extracts were incubated with antibodies specific against calsequestrin or the ATPase and immunoprecipitates were separated by disc gel electrophoresis. Incorporation of radioactivity into bands identified as calsequestrin or the ATPase was analyzed by counting of gel slices. In Dulbecco's modified Eagles medium (DME medium) containing 0.1 volume of horse serum and 0.005 volume of chick embryo extract, the cells began to fuse after about 50 h in culture, forming multinucleated myotubes. Calsequestrin synthesis was barely detectable after 24 h in culture. After 44 h, before fusion of myoblasts began, the rate of calsequestrin synthesis increased severalfold. The rate of synthesis continued to increase until about 72 h and then diminished. If cells were transferred at 44 h to DME medium containing 0.2 volume of fetal calf serum and 0.08 volume of chick embryo extract, fusion was delayed by about 20 h. In this medium the rate of calsequestrin synthesis diminished after a peak at 44 h but, by contrast, the rate of synthesis of the ATPase increased dramatically following fusion at about 80 h. If cells were transferred at about 40 h to DME medium containing 0.1 volume of horse serum and only 60 muM Ca2+ the cells did not fuse and, again, the rate of calsequestrin synthesis was diminished after a peak at about 40 h. By contrast the rate of ATPase synthesis increased sharply in spite of the lack of fusion. Both proteins were degraded with a half-life of about 20 h. These studies show that the synthesis of calsequestrin, an extrinsic membrane protein, and the ATPase, an intrinsic protein of the same membrane, are synthesized under separate control.

Adenosine Triphosphatases↗
PubMed1989

Plant cells contain calsequestrin.

Calsequestrin is a high capacity low affinity Ca2+-binding protein thought to be essential for the function of the intracellular rapid releasable Ca2+ pool of a variety of animal cells. Here we show that two types of plant tissues, cultured Streptanthus tortuosus cells and spinach leaves, contain a form of calsequestrin. In subcellular fractions of S. tortuosus cells, Stains-all staining reveals a metachromatically blue-staining 56,000-Da protein enriched in the microsomal fraction. This protein shares several biochemical characteristics with animal calsequestrin: 1) it changes its apparent molecular weight with the pH; 2) it is able to bind 45Ca2+ on nitrocellulose transfers; and 3) it is recognized by antibodies against canine cardiac calsequestrin. Calsequestrin was also identified in spinach leaves using a direct extraction procedure that was developed for muscle calsequestrin. Thus, our results demonstrate that plant cells contain calsequestrin within a subcellular membrane fraction. These results also suggest that calsequestrin is an ubiquitous protein rather than being limited only to animal cells.

Animals↗
PubMed1988

Complete amino acid sequence of canine cardiac calsequestrin deduced by cDNA cloning.

cDNA cloning was used to deduce the complete amino acid sequence of canine cardiac calsequestrin, the principal Ca2+-binding protein of cardiac junctional sarcoplasmic reticulum. Cardiac calsequestrin contains 391 amino acid residues plus a 19-residue amino-terminal signal sequence. The molecular weight of the mature protein, excluding carbohydrate, is 45,269. Cardiac calsequestrin is highly acidic, and a striking feature is the enrichment of acidic residues (60%) within the 63 carboxyl-terminal residues. No part of the sequence contains EF hand Ca2+-binding structures. The photo-affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine was used to localize the Ca2+-regulated hydrophobic site to amino acid residues 192-223. The cardiac and skeletal muscle isoforms of calsequestrin (Fliegel, L., Ohnishi, M., Carpenter, M. R., Khanna, V. K., Reithmeier, R. A. F., and MacLennan, D. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1167-1171), although the products of different genes, are 65% identical, are acidic, and share one glycosylation site. However, cardiac calsequestrin has several unique features. First, it has a 31-amino acid extension at its carboxyl terminus (residues 361-391), which contains 71% acidic residues and a second glycosylation site. Second, its mRNA contains a second open reading frame with the capacity to code for a 111-amino acid protein. Third, contrary to the restricted expression of the fast skeletal isoform, cardiac calsequestrin mRNA is present in both cardiac and slow skeletal muscle, but not in fast skeletal muscle. We conclude that the deduced amino acid sequence of cardiac calsequestrin is consistent with its ability to bind large amounts of Ca2+ (40 mol of Ca2+/mol of calsequestrin). The protein probably binds Ca2+ by acting as a charged surface rather than by presenting multiple discrete Ca2+-binding sites.

Amino Acid Sequence↗
PubMed1996

Regulation of calcium binding proteins calreticulin and calsequestrin during differentiation in the myogenic cell line L6.

In this report we defined the structural and temporal limits within which calreticulin and calsequestrin participate in the muscle cell phenotype, in the L6 model myogenic system. Calreticulin and calsequestrin are two Ca2+ binding proteins thought to participate in intracellular Ca2+ homeostasis. We show that calsequestrin protein and mRNA were expressed when L6 cells were induced to differentiate, during which time the level of expression of calreticulin protein did not change appreciably. Calreticulin mRNA levels, however, were constant throughout L6 cell differentiation except for slight decline in the mRNA levels at the very late stages of L6 differentiation (day 11-12). We also show that the two Ca2+ binding proteins are coexpressed in differentiated L6 cells. Based on its mobility in SDS-PAGE, L6 rat skeletal muscle cells in culture expressed cardiac isoform of calsequestrin. In the mature rat skeletal muscle, calreticulin and calsequestrin were localized to sarcoplasmic reticulum (SR). Calreticulun, but not calsequestrin, staining was also observed in the perinuclear region. These data suggest that expression of calreticulin and calsequestrin may be under different control during myogenesis in rat L6 cells in culture.

Animals↗
PubMed1986

Characteristics of skeletal muscle calsequestrin: comparison of mammalian, amphibian and avian muscles.

Calsequestrin was identified in the isolated sarcoplasmic reticulum from skeletal muscle of three mammalian species (man, rat and rabbit) and from frog and chicken muscle, using electrophoretic and immunoblot techniques. It was further characterized in sarcoplasmic reticulum protein mixtures and at several stages of purification, following extraction with EDTA. We found extensive similarities in apparent molecular weight values, Stains All staining properties and in Cleveland's peptide maps, between mammalian calsequestrins, and no detectable difference within a species between fast and slow muscle. Human calsequestrin, with an apparent molecular weight of 60,000 when measured at alkaline pH and of 41,000 when measured at neutral pH, appears to be the smallest in size. Frog calsequestrin, although weakly cross-reactive with rabbit calsequestrin and having a relatively higher apparent molecular weight at alkaline pH (72,000), shares several significant properties with mammalian calsequestrins. It bound calcium with a high capacity (1300 nmol per mg protein), it contained about 32% acidic amino acid residues and focused at closely similar pI values. We observed the formation of a complex with Stains All absorbing maximally at 535 nm, rather than at 600 nm, and an even more marked shift in apparent molecular weight at neutral pH. We found distinct differences in the case of chicken calsequestrin, in addition to those previously reported. It is a highly acidic, calcium-precipitable protein, but its amino acid composition is contradistinguished by a higher ratio of glutamate to aspartate and its rate of electrophoretic mobility is minimally affected by changes in pH. It stained deep bluish with Stains All after gel electrophoresis and yielded a protein-dye complex in aqueous solution, absorbing maximally at 560 nm, and finally, it bound fluorescent Concanavalin A.

Animals↗
PubMed2001

Structure and expression of the mouse cardiac calsequestrin gene.

Calsequestrin is a sarcoplasmic reticulum protein, which plays a predominant role in diastolic Ca2+-storage in the mammalian heart. The present study was designed to define the gene structure, developmental and tissue specific expression of the murine, cardiac isoform of calsequestrin. Two sets of genomic libraries (lambda phage and PAC) were screened using the mouse cardiac calsequestrin cDNA, and several overlapping clones were isolated. These clones were characterized using restriction enzyme digestion, Southern blotting and partial sequencing. The cardiac calsequestrin gene consists of 11 exons and its 5' flanking region is characterized by the presence of a TATA-like box, muscle specific promoter elements such as 7 E-boxes, 1 MEF-2, 1 MCBF and 1 Repeat (musS) motifs, as well as several muscle non-specific transcriptional elements (AP-2A, NRE1, NRE2, p53, Spel and TFI-IIA). Expression of the cardiac isoform of calsequestrin was first detected on day 11 pre-birth and approached adult levels by day 4 post-birth. Expression of cardiac calsequestrin was also detected in adult fast-twitch skeletal muscle, thyroid, testis and epididymis tissues. This genomic characterization of cardiac calsequestrin may form the basis for further evaluation of its regulatory role in Ca2+ homeostasis and contractility in the murine heart.

5' Untranslated Regions↗
PubMed1991

Altered properties of calsequestrin and the ryanodine receptor in the cardiac sarcoplasmic reticulum of hibernating mammals.

A novel isoform of calsequestrin was identified in sarcoplasmic reticulum vesicles from myocardial tissue of two species of hibernating ground squirrel. The protein was identified as calsequestrin by its cross-reactivity with antibodies raised against bovine cardiac calsequestrin, its pH-sensitive mobility in sodium dodecylsulphate-polyacrylamide gels, staining blue with the cationic carbocyanine dye 'Stains-All', binding peroxidase-conjugated concanavalin A, its endoglycosidase F sensitivity. Its NH2-terminal amino acid sequence is similar, but not identical, to that already determined for cardiac calsequestrin. Some of the biochemical properties of this protein distinguish it from the other mammalian isoforms. It has a unique electrophoretic mobility in both alkaline and neutral sodium dodecylsulphate-polyacrylamide gel electrophoresis, it appears to have a molecular weight approximately 7% greater than that of cardiac calsequestrin from other mammalian species, and its glycosylation pattern differs. This novel form of calsequestrin is expressed in cardiac SR vesicles which possess an abnormally high number of Ca2(+)-release channel/ryanodine receptor molecules. This ryanodine receptor also shows an altered Ca2(+)-sensitivity of ryanodine binding. The divergent biophysical properties of this novel form of cardiac calsequestrin, together with the apparently atypical ryanodine receptors in the cardiac sarcoplasmic reticulum membranes may have some functional significance in the adaptive mechanisms which allow the heart to function despite the severely reduced body temperatures (to approx. 0 degree C) encountered during hibernation.

Amino Acid Sequence↗
PubMed1989

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.

Animals↗
PubMed1983

A novel method for the isolation of calsequestrin from porcine skeletal muscle sarcoplasmic reticulum.

Calsequestrin was selectively solubilized from porcine skeletal muscle sarcoplasmic reticulum by incubation with HgCl2 and deoxycholate. While deoxycholate alone caused non-specific solubilization of calsequestrin and other extrinsic proteins, HgCl2 alone failed to extract significant amounts of extrinsic proteins. The simultaneous presence of HgCl2 and deoxycholate was necessary for the selective and quantitative solubilization of calsequestrin. Following DEAE-Sephadex ion-exchange chromatography of the HgCl2/ deoxycholate-solubilized material, calsequestrin was obtained in nearly homogeneous form. Porcine calsequestrin was similar to rabbit calsequestrin with respect to its amino acid composition and apparent molecular weight as determined by SDS-polyacrylamide gel electrophoresis. The calcium-binding capacity of porcine calsequestrin was 400 nmol of Ca2+ bound/mg protein, lower than the values reported for the rabbit protein.

Amino Acids↗
PubMed1994

Expression of muscle calsequestrin in epithelial HeLa cells: distribution and functional role.

Calsequestrin is a Ca2+ binding protein expressed by a few cell types (mostly muscle fibers). In these cells the distribution of the protein is within the endoplasmic/sarcoplasmic reticulum, however, not uniformly throughout but at discrete sites of the lumen. In order to investigate the mechanisms of this unusual intracellular distribution together with the possible functions of the protein, we have studied stable transfected clones of epithelial HeLa cells. Treatment of these cells with butyric acid induced a rapid (24 h) and massive (approx. 10-fold) increase of the transfected protein, whereas the other lumenal and membrane proteins of the endoplasmic reticulum were either modified slightly or unchanged. When butyric acid treatment was interrupted the calsequestrin levels returned rapidly (within 24 h) to the pre-treatment level. Such a rapid turnover was due in part to secretion, sustained by both spontaneous and Ca(2+)-dependent release of calsequestrin to the extracellular medium. From the physiological point of view, the transfected cells exhibited only moderate increases of the Ca2+ release responses triggered by either ATP (a ligand addressed to the P2u receptor and working through IP3 generation) or thapsigargin (a blocker of the endoplasmic reticulum Ca2+ ATPase), with no further increase after butyric acid induction of calsequestrin. This result appears to correlate with the occurrence of only small amounts of calsequestrin within the endoplasmic reticulum lumen of all transfected cells. The bulk of calsequestrin, in contrast, was found sequestered within large vacuoles distributed both near the cell surface and, after butyric acid treatment, also in the deep cytoplasm. These vacuoles (possibly a lysosomal subcompartment) appear to contain no Ca2+ as no difference in 45Ca release from transfected cells was observed without or with butyric acid pretreatment when exposed to ionomycin, alone or combined with monensin. We conclude that HeLa cells possess no adequate mechanisms to keep calsequestrin in its physiologically relevant location, the endoplasmic reticulum. In the transfected cell the protein seems therefore to be diverted (possibly by default) to vacuoles destined to be rapidly eliminated by the cell.

Butyrates↗