Sarcoplasmic reticulum calsequestrins: Structural and functional properties

Calsequestrin is the major Ca²⁺-binding protein localized in the terminal cisternae of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle cells. Calsequestrin has been purified and cloned from both skeletal and cardiac muscle in mammalian, amphibian, and avian species. Two different calsequestrin gene products namely cardiac and fast have been identified. Fast and cardiac calsequestrin isoforms have a highly acidic amino acid composition. The amino acid composition of the cardiac form is very similar to the skeletal form except for the carboxyl terminal region of the protein which possess variable length of acidic residues and two phosphorylation sites. Circular dichroism and NMR studies have shown that calsequestrin increases its -helical content and the intrinsic fluorescence upon binding of Ca²⁺. Calsequestrin binds Ca²⁺ with high-capacity and with moderate affinity and it functions as a Ca²⁺ storage protein in the lumen of the SR. Calsequestrin has been found to be associated with the Ca²⁺ release channel protein complex of the SR through protein-protein interactions. The human and rabbit fast calsequestrin genes have been cloned. The fast gene is skeletal muscle specific and transcribed at different rates in fast and slow skeletal muscle but not in cardiac muscle. We have recently cloned the rabbit cardiac calsequestrin gene. Heart expresses exclusively the cardiac calsquestrin gene. This gene is also expressed in slow skeletal muscle. No change in calsequestrin mRNA expression has been detected in animal models of cardiac hypertrophy and in failing human heart.     

Ca-ATPase activity and protein composition of sarcoplasmic reticulum membranes isolated from skeletal muscles of typical hibernator,the ground squirrel Spermophilus undulatus

Ca-ATPase activity in sarcoplasmic reticulum (SR) membranes isolated from skeletal muscles of the typical hibernator, the ground squirrel Spermophilus undulatus, is about 2-fold lower than that in SR membranes of rats and rabbits and is further decreased 2-fold during hibernation. The use of carbocyanine anionic dye Stains-All has revealed that Ca-binding proteins of SR membranes, histidine-rich Ca-binding protein and sarcalumenin, in ground squirrel, rat, and rabbit SR have different electrophoretic mobility corresponding to apparent molecular masses 165, 155, and 170 kDa and 130, 145, and 160 kDa, respectively; the electrophoretic mobility of calsequestrin (63 kDa) is the same in all preparations. The content of these Ca-binding proteins in SR membranes of the ground squirrels is decreased 3–4 fold and the content of 55, 30, and 22 kDa proteins is significantly increased during hibernation.     

Post-natal heart adaptation in a knock-in mouse model of calsequestrin 2-linked recessive catecholaminergic polymorphic ventricular tachycardia

Cardiac calsequestrin (CASQ2) contributes to intracellular Ca²⁺ homeostasis by virtue of its low-affinity/high-capacity Ca²⁺ binding properties, maintains sarcoplasmic reticulum (SR) architecture and regulates excitation–contraction coupling, especially or exclusively upon β-adrenergic stimulation. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease associated with cardiac arrest in children or young adults. Recessive CPVT variants are due to mutations in the CASQ2 gene. Molecular and ultra-structural properties were studied in hearts of CASQ2^R33Q/R33Q and of CASQ2^−/− mice from post-natal day 2 to week 8. The drastic reduction of CASQ2-R33Q is an early developmental event and is accompanied by down-regulation of triadin and junctin, and morphological changes of jSR and of SR-transverse-tubule junctions. Although endoplasmic reticulum stress is activated, no signs of either apoptosis or autophagy are detected. The other model of recessive CPVT, the CASQ2^−/− mouse, does not display the same adaptive pattern. Expression of CASQ2-R33Q influences molecular and ultra-structural heart development; post-natal, adaptive changes appear capable of ensuring until adulthood a new pathophysiological equilibrium.     

An improved method for the isolation of rat cardiac sarcoplasmic reticulum

Summary Preparations of cardiac sarcoplasmic reticulum (CSR) isolated from the rat by differential centrifugation have been widely used for measuring alterations in intracellular calcium flux in response to metabolic and pharmacologic disruptions. However, the purity of these SR fractions has not been firmly established.Using a combination of differential and linear sucrose gradient centrifugation, we have isolated rat CSR with high specific activity and purity. By SDS-PAGE analysis, the preparation is enriched in a protein (110 kD) of similar size to the Ca²⁺-ATPase of SR from other sources. Gels stained with the dye Stains All reveal a blue colored 55 kD band, confirming the presence of calsequestrin, the intraluminal low-affinity calcium binding protein of SR. The presence of the transmembrane 53 kD glycoprotein of SR was confirmed by endoglycosidase-H treatment followed by SDS-PAGE and also by a modified Western blotting technique. The rate of calcium uptake in this preparation averages 130 nmol/mg over the first minute of accumulation, approximately 4 times that previously reported for rat CSR. Calcium uptake in our preparation was essentially complete within 5 minutes. Preparations isolated by this method should be of value in future studies measuring alterations in rat CSR function.     

Ryanodine receptor mutations in arrhythmia: The continuing mystery of channel dysfunction

Mutations in RyR2 are causative of an inherited disorder which often results in sudden cardiac death. Dysfunctional channel behaviour has been the subject of many investigations varying from single channel analysis through to complex animal models. This review discusses recent advances in the field, describes the controversy surrounding the exact consequences of RyR2 mutation and how the disparate data may be reconciled. This heterogeneity of function with respect to the effects of polymorphisms, phosphorylation, cytosolic and luminal Ca²⁺ as well as inter-domain interactions may have important implications for the recent pharmaceutical therapies which have been put forward. We surmise that a comprehensive characterisation of mutations on a case-by-case basis may be beneficial for the development of specifically targeted therapies.     

Calcium binding proteins in the sarcoplasmic/endoplasmic reticulum of muscle and nonmuscle cells

In this paper we review some of the large quantities of information currently available concerning the identification, structure and function of Ca²⁺-binding proteins of endoplasmic and sarcoplasmic reticulum membranes. The review places particular emphasis on identification and discussion of Ca²⁺ storage proteins in these membranes. We believe that the evidence reviewed here supports the contention that the Ca²⁺-binding capacity of both calsequestrin and calreticulin favor their contribution as the major Ca²⁺-binding proteins of muscle and nonmuscle cells, respectively. Other Ca²⁺-binding proteins discovered in both endoplasmic reticulum and sarcoplasmic reticulum membranes probably contribute to the overall Ca²⁺ storage capacity of these membrane organelles, and they also play other important functional role such as posttranslational modification of newly synthesized proteins, a cytoskeletal (structural) function, or movement of Ca²⁺ within the lumen of the sarcoplasmic/endoplasmic reticulum towards the storage sites.Abbreviations SR Sarcoplasmic Reticulum - ER Endoplasmic Reticulum - InsP₃ Inositol 1,4,5-trisphosphate - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - PDI Protein Disulphide Isomerase - T₃BP Thyroid Hormone Binding Protein - Grp Glucose regulated proteins - HCP Histidine-rich Ca²⁺ binding Protein - LDL Low Density Lipoprotein     

Characteristics of sarcoplasmic reticulum membrane preparations isolated from skeletal muscles of active and hibernating ground squirrel Spermophilus undulatus

The total Ca-ATPase activity in the sarcoplasmic reticulum (SR) membrane fraction isolated from skeletal muscles of winter hibernating ground squirrel Spermophilus undulatus is 2.2-fold lower than in preparations obtained from summer active animals. This is connected in part with 10% decrease of the content of Ca-ATPase protein in SR membranes. However, the enzyme specific activity calculated with correction for its content in SR preparations is still 2-fold lower in hibernating animals. Analysis of the protein composition of SR membranes has shown that in addition to the decrease in Ca-ATPase content in hibernating animals, the amount of SR Ca-release channel (ryanodine receptor) is decreased 2-fold, content of Ca-binding proteins calsequestrin, sarcalumenin, and histidine-rich Ca-binding protein is decreased 3-4-fold, and the amount of proteins with molecular masses 55, 30, and 22 kD is significantly increased. Using the cross-linking agent cupric–phenanthroline, it was shown that in SR membranes of hibernating ground squirrels Ca-ATPase is present in a more aggregated state. The affinity of SR membranes to the hydrophilic fluorescent probe ANS is higher and the degree of excimerization of the hydrophobic probe pyrene is lower (especially for annular lipids) in preparations from hibernating than from summer active animals. The latter indicates an increase in the microviscosity of the lipid environment of Ca-ATPase during hibernation. We suggest that protein aggregation as well as the changes in protein composition and/or in properties of lipid bilayer SR membranes can result in the decrease of enzyme activity during hibernation.     

Cross-linking of the sarcoplasmic reticulum ATPase protein.

Treatment of rabbit sarcoplasmic reticulum vesicles with the cross-linking agent, cupric phenanthroline, causes production of high-molecular weight bands on SDS-gel electrophoresis. A plot of log mol wt $\text{vs}$ mobility indicates that the main band produced from the ATPase (mol wt = 10⁵) has a mol wt of 4 × 10⁵ and thus suggests formation of a tetramer. Notably, bands corresponding to dimers, trimers, pentamers, etc., are absent. The bands attributable to calsequestrin and calcium binding protein are unchanged by cupric phenanthroline. With extended treatment, the tetramer itself is polymerized (mol wt>10⁶). Partial disruption of the membranes with deoxycholate or Triton X-100 before cross-linking favors tetramer formation; the presence of sodium dodecyl sulfate, on the other hand, prevents intermolecular cross-linking. Our results suggest that the ATPase is at least partially associated within the membrane as a tetramer.     

Proteomic profiling of naturally protected extraocular muscles from the dystrophin-deficient mdx mouse

Duchenne muscular dystrophy is the most frequent neuromuscular disorder of childhood. Although this x-linked muscle disease is extremely progressive, not all subtypes of skeletal muscles are affected in the same way. While extremities and trunk muscles are drastically weakened, extraocular muscles are usually spared in Duchenne patients. In order to determine the global protein expression pattern in these naturally protected muscles we have performed a comparative proteomic study of the established mdx mouse model of x-linked muscular dystrophy. Fluorescence difference in-gel electrophoretic analysis of 9-week-old dystrophin-deficient versus age-matched normal extraocular muscle, using a pH 4-7 gel range, identified out of 1088 recognized protein spots a moderate expression change in only seven protein species. Desmin, apolipoprotein A-I binding protein and perilipin-3 were found to be increased and gelsolin, gephyrin, transaldolase, and acyl-CoA dehydrogenase were shown to be decreased in mdx extraocular muscles. Immunoblotting revealed a drastic up-regulation of utrophin, comparable levels of β-dystroglycan and key Ca²⁺-regulatory elements, and an elevated concentration of small stress proteins in mdx extraocular muscles. This suggests that despite the lack of dystrophin only a limited number of cellular systems are perturbed in mdx extraocular muscles, probably due to the substitution of dystrophin by its autosomal homolog. Utrophin appears to prevent the loss of dystrophin-associated proteins and Ca²⁺-handling elements in extraocular muscle tissue. Interestingly, the adaptive mechanisms that cause the sparing of extraocular fibers seem to be closely linked to an enhanced cellular stress response.