Characterization of junctate-SERCA2a interaction in murine cardiomyocyte

Junctate is a newly identified sarcoplasmic reticulum (SR) Ca²⁺ binding protein, but its function in cardiac muscle has remained unclear. Our previous study showed that chronic over-expression of junctate in transgenic mice led to altered SR functions and development of severe hypertrophy. In this study, we identified the interaction of junctate with SERCA2a by co-immunoprecipitation and GST-pull-down assay. This interaction was inhibited by higher Ca²⁺ concentration. Immunolocalization assays also showed that junctate and SERCA2a were co-localized in the SR of cardiomyocytes. Direct binding of the C-terminal region of junctate (amino acids 79-270) and luminal domain of SERCA2a (amino acids 70-89) was observed by deletion mutation experiments. Adenovirus-mediated transient over-expression of junctate in cardiomyocytes showed a reduced decay time of Ca²⁺ transients and increased oxalate-supported SERCA2 Ca²⁺ uptake, suggesting an increased activity of SERCA2a. Taken together, according to our data, junctate may play an important role in the regulation of SR Ca²⁺ cycling through the interaction with SERCA2a in the murine heart.     

Molecular cloning, expression, functional characterization, chromosomal localization, and gene structure of junctate, a novel integral calcium binding protein of sarco(endo)plasmic reticulum membrane

Screening a cDNA library from human skeletal muscle and cardiac muscle with a cDNA probe derived from junctin led to the isolation of two groups of cDNA clones. The first group displayed a deduced amino acid sequence that is 84% identical to that of dog heart junctin, whereas the second group had a single open reading frame that encoded a polypeptide with a predicted mass of 33 kDa, whose first 78 NH(2)-terminal residues are identical to junctin whereas its COOH terminus domain is identical to aspartyl beta-hydroxylase, a member of the alpha-ketoglutarate-dependent dioxygenase family. We named the latter amino acid sequence junctate. Northern blot analysis indicates that junctate is expressed in a variety of human tissues including heart, pancreas, brain, lung, liver, kidney, and skeletal muscle. Fluorescence in situ hybridization analysis revealed that the genetic loci of junctin and junctate map to the same cytogenetic band on human chromosome 8. Analysis of intron/exon boundaries of the genomic BAC clones demonstrate that junctin, junctate, and aspartyl beta-hydroxylase result from alternative splicing of the same gene. The predicted lumenal portion of junctate is enriched in negatively charged residues and is able to bind calcium. Scatchard analysis of equilibrium (45)Ca(2+) binding in the presence of a physiological concentration of KCl demonstrate that junctate binds 21.0 mol of Ca(2+)/mol protein with a k(D) of 217 +/- 20 microm (n = 5). Tagging recombinant junctate with green fluorescent protein and expressing the chimeric polypeptide in COS-7-transfected cells indicates that junctate is located in endoplasmic reticulum membranes and that its presence increases the peak amplitude and transient calcium released by activation of surface membrane receptors coupled to InsP(3) receptor activation. Our study shows that alternative splicing of the same gene generates the following functionally distinct proteins: an enzyme (aspartyl beta-hydroxylase), a structural protein of SR (junctin), and a membrane-bound calcium binding protein (junctate).     

Impaired Ca2+ store functions in skeletal and cardiac muscle cells from sarcalumenin-deficient mice

Sarcalumenin (SAR), specifically expressed in striated muscle cells, is a Ca2+-binding protein localized in the sarcoplasmic reticulum (SR) of the intracellular Ca2+ store. By generating SAR-deficient mice, we herein examined its physiological role. The mutant mice were apparently normal in growth, health, and reproduction, indicating that SAR is not essential for fundamental muscle functions. SAR-deficient skeletal muscle carrying irregular SR ultrastructures retained normal force generation but showed slow relaxation phases after contractions. A weakened Ca2+ uptake activity was detected in the SR prepared from mutant muscle, indicating that SAR contributes to Ca2+ buffering in the SR lumen and also to the maintenance of Ca2+ pump proteins. Cardiac myocytes from SAR-deficient mice showed slow contraction and relaxation accompanied by impaired Ca2+ transients, and the mutant mice exhibited a number of impairments in cardiac performance as determined in electrocardiography, ventricular catheterization, and echocardiography. The results obtained demonstrate that SAR plays important roles in improving the Ca2+ handling functions of the SR in striated muscle.     

Molecular architecture of Ca2+ signaling control in muscle and heart cells

Ca2+ signaling in skeletal and cardiac muscles is a bi-directional process that involves cross-talk between signaling molecules in the sarcolemmal membrane and Ca2+ release machinery in the intracellular organelles. Maintenance of a junctional membrane structure between the sarcolemmal membrane and the sarcoplasmic reticulum (SR) provides a framework for the conversion of action potential arrived at the sarcolemma into release of Ca2+ from the SR, leading to activation of a variety of physiological processes. Activity-dependent changes in Ca2+ storage inside the SR provides a retrograde signal for the activation of store-operated Ca2+ channel (SOC) on the sarcolemmal membrane, which plays important roles in the maintenance of Ca2+ homeostasis in physiology and pathophysiology. Research progress during the last 30 years had advanced our understanding of the cellular and molecular mechanisms for the control of Ca2+ signaling in muscle and cardiovascular physiology. Here we summarize the functions of three key molecules that are located in the junctional membrane complex of skeletal and cardiac muscle cells: junctophilin as a "glue" that physiologically links the SR membrane to the sarcolemmal membrane for formation of the junctional membrane framework, mitsugumin29 as a muscle-specific synaptophysin family protein that contributes to maintain the coordinated Ca2+ signaling in skeletal muscle, and TRIC as a novel cation-selective channel located on the SR membrane that provides counter-ion current during the rapid process of Ca2+ release from the SR.     

Uncoupling store-operated Ca2+ entry and altered Ca2+ release from sarcoplasmic reticulum through silencing of junctophilin genes

Junctophilin (JP) mediates the close contact between cell surface and intracellular membranes in muscle cells ensuring efficient excitation-contraction coupling. Here we demonstrate that disruption of triad junction structure formed by the transverse tubular (TT) invagination of plasma membrane and terminal cisternae of sarcoplasmic reticulum (SR) by reduction of JP expression leads to defective Ca2+ homeostasis in muscle cells. Using adenovirus with small hairpin interference RNA (shRNA) against both JP1 and JP2 genes, we could achieve acute suppression of JPs in skeletal muscle fibers. The shRNA-treated muscles exhibit deformed triad junctions and reduced store-operated Ca2+ entry (SOCE), which is likely due to uncoupled retrograde signaling from SR to TT. Knockdown of JP also causes a reduction in SR Ca2+ storage and altered caffeine-induced Ca2+ release, suggesting an orthograde regulation of the TT membrane on the SR Ca2+ release machinery. Our data demonstrate that JPs play an important role in controlling overall intracellular Ca2+ homeostasis in muscle cells. We speculate that altered expression of JPs may underlie some of the phenotypic changes associated with certain muscle diseases and aging.     

Abnormal sarcoplasmic reticulum ryanodine receptor in malignant hyperthermia

Previous studies have demonstrated that skeletal muscle from individuals susceptible to malignant hyperthermia (MH) has a defect associated with the mechanism of calcium release from its intracellular storage sites in the sarcoplasmic reticulum (SR). In this report we demonstrate that the [3H]ryanodine receptor of isolated MH-susceptible (MHS) porcine heavy SR exhibits an altered Ca2+ dependence of [3H]ryanodine binding at the low affinity Ca2+ site as well as a lower Kd for ryanodine (92 versus 265 nM) when compared to normal porcine SR. The Bmax of the normal and MHS [3H] ryanodine receptor (9.3-12.6 pmol/mg) was not significantly different, and analysis of MHS and normal SR proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis did not reveal a significant difference in the intensity of Coomassie Blue staining of the spanning protein/ryanodine receptor region of the gels (Mr greater than 300,000). We also find that MHS porcine muscle intact fiber bundles exhibit a 5-10-fold lower ryanodine threshold for twitch and tetanus inhibition, and contracture onset when compared to normal muscle. Since the SR ryanodine receptor is a calcium release channel as well as a component intimately involved in transverse tubule-SR communication, abnormalities in the skeletal muscle ryanodine receptor may be responsible for the abnormal SR calcium release and contractile properties demonstrated by MHS muscle.     

Retrograde regulation of store-operated calcium channels by the ryanodine receptor-associated protein triadin 95 in rat skeletal myotubes

The 95kDa triadin (or T95), the main skeletal muscle triadin isoform, negatively regulates the mechanism of excitation-contraction coupling. T95 is a ryanodine receptor (RyR)-interacting protein but it also possesses a calsequestrin-interacting domain. RyR and calsequestrin are involved in Ca2+ signalling and, for instance, influence the activity of store-dependent Ca2+ channels (SOC). This work was undertaken to determine whether T95 was able to modulate the entry of Ca2+ through SOC. The experiments were carried out on differentiated rat myotubes over-expressing T95 or DsRed (control cells) by means of an adenovirus infection. Intracellular Ca2+ signals were analyzed using the Ca2+ indicator Fluo-4. The sarco-endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin was used to deplete intracellular Ca2+ stores. When applied in the presence of a Ca2+-free medium, thapsigargin elicited transient but long-lasting Fluo-4 responses by elevating the cytoplasmic concentration of Ca2+ ([Ca2+]i). The over-expression of T95 reduced the thapsigargin-dependent [Ca2+]i increase, with respect to control myotubes. Addition of extracellular Ca2+after the depletion of this Ca2+ pool was accompanied by a [Ca2+]i increase that was sensitive to the SOC blockers 2-APB, SKF-96365 and La3+. The over-expression of T95 reduced this Ca2+ influx, without changing its pharmacological properties, showing that T95 over-expression did not alter the properties of the SOC. In conclusion, the RyR-interacting molecule T95, recently shown to inhibit the excitation-contraction coupling, has also the ability to interfere with the skeletal muscle Ca2+ signalling by depressing thapsigargin-dependent Ca2+ release and influx.     

Drug action of thapsigargin on the Ca2+ pump protein of sarcoplasmic reticulum.

Thapsigargin is found to be a potent inhibitor of the intracellular Ca2+ pump proteins from skeletal muscle sarcoplasmic reticulum (SR), cardiac SR, and brain microsomes. For skeletal muscle SR, the molar ratio of thapsigargin to Ca2+ pump protein for complete inhibition (MRc) of the Ca2+ loading rate, Ca(2+)-dependent ATPase activity, and formation of phosphorylated intermediate (EP) was approximately 1. When the Ca2+ pump protein of low affinity to Ca2+ (E2 state) was pretreated with thapsigargin, ATP and Ca2+ binding to the Ca2+ pump protein was completely inhibited. In the presence of Ca2+ (E1 state), Ca2+ pump protein was protected from inactivation by thapsigargin with respect to Ca2+ binding and EP formation. The MRc for brain microsomes, which mediate Ca2+ uptake into intracellular (inositol 1,4,5-trisphosphate-releasable) Ca2+ pools, is likewise stoichiometric. Approximately 30% of Ca2+ loading activity of brain microsomes was insensitive to thapsigargin, indicating the presence of other Ca2+ pumping system(s). The MRc for heart is 3.8, indicating that the Ca2+ pump of cardiac SR is less sensitive to thapsigargin. Phosphorylation of cardiac SR with protein kinase A increased the sensitivity to thapsigargin to MRc of 2.8. In summary, we find that: 1) thapsigargin is the most effective inhibitor of the Ca2+ pump protein of intracellular membranes (SR and endoplasmic reticulum); 2) its primary inhibitory action appears to inactivate the E2 form of the enzyme preferentially; 3) cardiac SR shows lesser sensitivity to thapsigargin than skeletal muscle SR and brain microsomes; protein kinase A treatment of cardiac SR enhances the sensitivity to the drug.     

A novel interaction mechanism accounting for different acylphosphatase effects on cardiac and fast twitch skeletal muscle sarcoplasmic reticulum calcium pumps

In cardiac and skeletal muscle Ca2+ translocation from cytoplasm into sarcoplasmic reticulum (SR) is accomplished by different Ca2+-ATPases whose functioning involves the formation and decomposition of an acylphosphorylated phosphoenzyme intermediate (EP). In this study we found that acylphosphatase, an enzyme well represented in muscular tissues and which actively hydrolyzes EP, had different effects on heart (SERCA2a) and fast twitch skeletal muscle SR Ca2+-ATPase (SERCA1). With physiological acylphosphatase concentrations SERCA2a exhibited a parallel increase in the rates of both ATP hydrolysis and Ca2+ transport; in contrast, SERCA1 appeared to be uncoupled since the stimulation of ATP hydrolysis matched an inhibition of Ca2+ pump. These different effects probably depend on phospholamban, which is associated with SERCA2a but not SERCA1. Consistent with this view, the present study suggests that acylphosphatase-induced stimulation of SERCA2a, in addition to an enhanced EP hydrolysis, may be due to a displacement of phospholamban, thus to a removal of its inhibitory effect.     

Halothane modulation of skeletal muscle ryanodine receptors: dependence on Ca2+, Mg2+, and ATP

Malignant hyperthermia (MH) susceptibility is a genetic disorder of skeletal muscle associated with mutations in the ryanodine receptor isoform 1 (RyR1) of sarcoplasmic reticulum (SR). In MH-susceptible skeletal fibers, RyR1-mediated Ca(2+) release is highly sensitive to activation by the volatile anesthetic halothane. Indeed, studies with isolated RyR1 channels (using simple Cs(+) solutions) found that halothane selectively affects mutated but not wild-type RyR1 function. However, studies in skeletal fibers indicate that halothane can also activate wild-type RyR1-mediated Ca(2+) release. We hypothesized that endogenous RyR1 agonists (ATP, lumenal Ca(2+)) may increase RyR1 sensitivity to halothane. Consequently, we studied how these agonists affect halothane action on rabbit skeletal RyR1 reconstituted into planar lipid bilayers. We found that cytosolic ATP is required for halothane-induced activation of the skeletal RyR1. Unlike RyR1, cardiac RyR2 (much less sensitive to ATP) responded to halothane even in the absence of this agonist. ATP-dependent halothane activation of RyR1 was enhanced by cytosolic Ca(2+) (channel agonist) and counteracted by Mg(2+) (channel inhibitor). Dantrolene, a muscle relaxant used to treat MH episodes, did not affect RyR1 or RyR2 basal activity and did not interfere with halothane-induced activation. Studies with skeletal SR microsomes confirmed that halothane-induced RyR1-mediated SR Ca(2+) release is enhanced by high ATP-low Mg(2+) in the cytosol and by increased SR Ca(2+) load. Thus, physiological or pathological processes that induce changes in cellular levels of these modulators could affect RyR1 sensitivity to halothane in skeletal fibers, including the outcome of halothane-induced contracture tests used to diagnose MH susceptibility.     

Intracellular EDTA mimics parvalbumin in the promotion of skeletal muscle relaxation

Parvalbumin (PA) is an intracellular Ca2+-binding protein found in some muscle and nerves. Its ability to bind Ca2+ and facilitate skeletal muscle relaxation is limited by its Mg2+ off-rate. EDTA serves as an "artificial" PA in that it exhibited similar rate constants for Mg2+ (3 s-1) and Ca2+ (0.7 s-1) dissociation at 10 degrees C. When introduced into frog skeletal muscle, EDTA increased the relaxation rate by approximately 2.7-fold, and with increasing tetanus duration, EDTA lost its ability to contribute to relaxation (and Ca2+ sequestration) at its Mg2+ off-rate. Intracellular EDTA recovered its ability to contribute to muscle relaxation and Ca2+ sequestration at its Ca2+ off-rate. Like PA, EDTA's contribution to muscle relaxation and Ca2+ sequestration was more clearly observed when the SR Ca-ATPase was inhibited. Introduction of EDTA into rat soleus muscle, which has low [PA], increased the relaxation rate in a manner that was analogous to the way in which PA facilitates relaxation of frog skeletal muscle. Thus intracellular EDTA serves as an effective mimic of PA, and its use should aid in our understanding of PA's function in muscle and nerve.     

Physiological significance of Ca uptake by mitochondria in the heart in comparison with that by cardiac sarcoplasmic reticulum.

It was investigated whether mitochondria play a significant role in the physiological regulation of the contractile process by Ca2+ in cardiac muscle in comparison with the sarcoplasmic reticulum (SR). Ca uptake activities of chicken cardiac SR and rabbit cardiac mitochondria were measured by means of centrifugation, dual-wave-length spectrophotometric and Millipore filtration methods. The maximum Ca uptake capacity of cardiac SR was usually 50-60 nmoles/mg protein and the apparent binding constant was 2.0 X 10(6) M-1. The apparent Ca-binding constant of cardiac mitochondria under limited loading conditions was 2.4 X 10(5) M-1 at pH 7.4 and 5.9 X 10(4) M-1 at pH 6.8. In the presence of 100 muM Ca2+ at 28-29 degrees, the estimated initial rate of Ca uptake of cardiac SR ranged from 20 to 30 nmoles Ca/mg-sec, while that of mitochondria was 4.6 nmoles Ca/mg-sec under limited loading conditions at pH 7.4 and 0.64 nmoles Ca/mg-sec under massive loading conditions at pH 6.8, which was much closer to physiological conditions. In the presence of low Ca2+ concentrations, the initial rate of Ca uptake of cardiac SR was 0.5 nmoles Ca/mg-sec at 3.5 X 10(-7) M Ca2+ and that of mitochondria under massive loading conditions at 1 X 10(-6) M Ca2+ was 0.02 nmoles Ca/mg-sec at pH 7.4 and 0.004 nmoles Ca/mg-sec at pH 6.8. The Ca uptake activities were also examined using glycerol-extracted cardiac muscle fibers. Cardiac SR, 1.7 mg/ml, reduced the tension of maximally contracted cardiac muscle fibers to a level corresponding to about 30% of maximum tension, but in the presence of 14.3 mg/ml of mitochondria the maximum tensions of both skeletal muscle and cardiac muscle fibers were maintained for at least 3 min. From these results the time course of relaxation of cardiac muscle induced by cardiac SR or mitochondria was calculated. It was concluded that, in the physiological contraction of cardiac muscle, the SR plays a major role in controlling intracellular Ca2+ movement; the Ca uptake of mitochondria is relatively insignificant. When the cardiac muscle contracts maximally, SR alone cannot relax the cardiac muscle without the aid of other Ca removing system.     

The changes in Ca2+ sparks associated with measured modifications of intra-store Ca2+ concentration in skeletal muscle

In cardiac muscle and amphibian skeletal muscle, the intracellular Ca2+ release that signals contractile activation proceeds by discrete local packets, which result in Ca2+ sparks. The remarkably stereotyped duration of these release events requires a robustly timed termination mechanism. In cardiac muscle the mechanism of spark termination appears to crucially involve depletion of Ca2+ in the lumen of the sarcoplasmic reticulum (SR), but in skeletal muscle, the mechanism is unknown. We used SEER (shifted excitation and emission ratioing of fluorescence) of SR-trapped mag-indo-1 and confocal imaging of fluorescence of cytosolic rhod-2 to image Ca2+ sparks while reversibly changing and measuring [Ca2+] in the SR ([Ca2+]SR) of membrane-permeabilized frog skeletal muscle cells. Sparks were collected in cells immersed in a solution promoting production of events at moderate frequency. Just after permeabilization, event frequency was zero, and in 10 minutes it reached close to a steady value. Controlled interventions modified [Ca2+]SR reversibly between a low value (299 microM on average in 10 experiments) and a high value (433 microM, a 45% average increase). This change increased sparks frequency by 93%, spatial width by 7%, rise time by 10%, and peak amplitude by 38% (provided that it was calculated in absolute terms, rather than normalized by resting fluorescence). The changes in event frequency and amplitude were statistically significant. The "strength" of the effect of [Ca2+]SR on frequency, quantified by decomposition of variance, was <6%. While the average change in [Ca2+]SR was limited, it reached up to 200% in individual fibers, without causing massive Ca2+ release or an increase of >3.5-fold in event frequency. Taken together with existing evidence that depletion is modest during Ca2+ sparks or release elicited by an action potential, the mild effects of [Ca2+]SR reported here do not support a major role of depletion in either the termination of sparks or the strong inactivation that terminates Ca2+ release at the global level in frog skeletal muscle.     

Transfer and tunneling of Ca2+ from sarcoplasmic reticulum to mitochondria in skeletal muscle

The role of mitochondrial Ca2+ transport in regulating intracellular Ca2+ signaling and mitochondrial enzymes involved in energy metabolism is widely recognized in many tissues. However, the ability of skeletal muscle mitochondria to sequester Ca2+ released from the sarcoplasmic reticulum (SR) during the muscle contraction-relaxation cycle is still disputed. To assess the functional cross-talk of Ca2+ between SR and mitochondria, we examined the mutual relationship connecting cytosolic and mitochondrial Ca2+ dynamics in permeabilized skeletal muscle fibers. Cytosolic and mitochondrial Ca2+ transients were recorded with digital photometry and confocal microscopy using fura-2 and mag-rhod-2, respectively. In the presence of 0.5 mM slow Ca2+ buffer (EGTA (ethylene glycolbis(2-aminoethylether)-N,N,N',N'-tetraacetic acid)), application of caffeine induced a synchronized increase in both cytosolic and mitochondrial [Ca2+]. 5 mM fast Ca2+ buffer (BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)) nearly eliminated caffeine-induced increases in [Ca2+]c but only partially decreased the amplitude of mitochondrial Ca2+ transients. Confocal imaging revealed that in EGTA, almost all mitochondria picked up Ca2+ released from the SR by caffeine, whereas only about 70% of mitochondria did so in BAPTA. Taken together, these results indicated that a subpopulation of mitochondria is in close functional and presumably structural proximity to the SR, giving rise to subcellular microdomains in which Ca2+ has preferential access to the juxtaposed organelles.     

Deficiency of triad junction and contraction in mutant skeletal muscle lacking junctophilin type 1

In skeletal muscle excitation-contraction (E-C) coupling, the depolarization signal is converted from the intracellular Ca2+ store into Ca2+ release by functional coupling between the cell surface voltage sensor and the Ca2+ release channel on the sarcoplasmic reticulum (SR). The signal conversion occurs in the junctional membrane complex known as the triad junction, where the invaginated plasma membrane called the transverse-tubule (T-tubule) is pinched from both sides by SR membranes. Previous studies have suggested that junctophilins (JPs) contribute to the formation of the junctional membrane complexes by spanning the intracellular store membrane and interacting with the plasma membrane (PM) in excitable cells. Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle. To examine the physiological role of JP-1 in skeletal muscle, we generated mutant mice lacking JP-1. The JP-1 knockout mice showed no milk suckling and died shortly after birth. Ultrastructural analysis demonstrated that triad junctions were reduced in number, and that the SR was often structurally abnormal in the skeletal muscles of the mutant mice. The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+. Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E-C coupling in skeletal muscle.     

The C terminus (amino acids 75-94) and the linker region (amino acids 42-54) of the Ca2+-binding protein S100A1 differentially enhance sarcoplasmic Ca2+ release in murine skinned skeletal muscle fibers

S100A1, a Ca2+-binding protein of the EF-hand type, is most highly expressed in striated muscle and has previously been shown to interact with the skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel/ryanodine receptor (RyR1) isoform. However, it was unclear whether S100A1/RyR1 interaction could modulate SR Ca2+ handling and contractile properties in skeletal muscle fibers. Since S100A1 protein is differentially expressed in fast- and slow-twitch skeletal muscle, we used saponin-skinned murine Musculus extensor digitorum longus (EDL) and Musculus soleus (Soleus) fibers to assess the impact of S100A1 protein on SR Ca2+ release and isometric twitch force in functionally intact permeabilized muscle fibers. S100A1 equally enhanced caffeine-induced SR Ca2+ release and Ca2+-induced isometric force transients in both muscle preparations in a dose-dependent manner. Introducing a synthetic S100A1 peptide model (devoid of EF-hand Ca2+-binding sites) allowed identification of the S100A1 C terminus (amino acids 75-94) and hinge region (amino acids 42-54) to differentially enhance SR Ca2+ release with a nearly 3-fold higher activity of the C terminus. These effects were exclusively based on enhanced SR Ca2+ release as S100A1 influenced neither SR Ca2+ uptake nor myofilament Ca2+ sensitivity/cooperativity in our experimental setting. In conclusion, our study shows for the first time that S100A1 augments contractile performance both of fast- and slow-twitch skeletal muscle fibers based on enhanced SR Ca2+ efflux at least mediated by the C terminus of S100A1 protein. Thus, our data suggest that S100A1 may serve as an endogenous enhancer of SR Ca2+ release and might therefore be of physiological relevance in the process of excitation-contraction coupling in skeletal muscle.     

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.     

Calcium transport by sarcoplasmic reticulum of skeletal muscle is inhibited by antibodies against the 53-kilodalton glycoprotein of the sarcoplasmic reticulum membrane

The effects of an antiserum against the 53-kDa glycoprotein (GP-53) of the sarcoplasmic reticulum (SR) and of monoclonal antibodies against GP-53 on Ca2+ transport and ATP hydrolysis by SR of rabbit skeletal muscle have been investigated. Preincubation of SR with an antiserum against GP-53 resulted in decreased ATP-driven Ca2+ transport by the SR but had no effect on Ca2+-stimulated ATP hydrolysis. Preincubation of SR with preimmune serum had no significant effect on either Ca2+ transport or Ca2+-ATPase activity. The effect of anti-GP-53 serum was time and concentration dependent. Preincubation of SR with two monoclonal antibodies against GP-53 had no effect on Ca2+ transport or on Ca2+-stimulated ATP hydrolysis. However, preincubation of SR with either monoclonal antibody against GP-53 together with a monoclonal antibody against the Ca2+-ATPase (at levels which had little effect alone) resulted in markedly decreased rates of Ca2+ uptake and ATP hydrolysis. Preincubation of SR with anti-GP-53-serum or with monoclonal antibodies, under the same conditions that inhibited Ca2+ uptake, did not increase the passive permeability of the SR membrane to Ca2+, did not decrease the permeability of the SR to oxalate, and did not cause significant proteolysis of the Ca2+-ATPase. Our results are consistent with the interpretation that GP-53 may modulate the function of the Ca2+-ATPase of the SR membrane.     

Ryanodine binding to sarcoplasmic reticulum membrane; comparison between cardiac and skeletal muscle

[3H]Ryanodine binding to skeletal muscle and cardiac sarcoplasmic reticulum (SR) vesicles was compared under experimental conditions known to inhibit or stimulate Ca2+ release. In the skeletal muscle SR, ryanodine binds to a single class of high-affinity sites (Kd of 11.3 nM). In cardiac SR vesicles, more than one class of binding sites is observed (Kd values of 3.6 and 28.1 nM). Ryanodine binding to skeletal muscle SR vesicles requires high concentrations of NaCl, whereas binding of the drug to cardiac SR is only slightly influenced by ionic strength. In the presence of 5'-adenylyl imidodiphosphate (p[NH]ppA), increased pH, and micromolar concentration of Ca2+ (which all induce Ca2+ release from SR) binding of ryanodine to SR is significantly increased in skeletal muscle, while being unchanged in cardiac muscle. Ryanodine binding to skeletal but not to cardiac muscle SR is inhibited in the presence of high Ca2+ or Mg2+ concentrations (all known to inhibit Ca2+ release from skeletal muscle SR). Ruthenium red or dicyclohexylcarbodiimide modification of cardiac and skeletal muscle SR inhibit Ca2+ release and ryanodine binding in both skeletal and cardiac membranes. These results indicate that significant differences exist in the properties of ryanodine binding to skeletal or cardiac muscle SR. Our data suggest that ryanodine binds preferably to site(s) which are accessible only when the Ca2+ release channel is in the open state.     

Ethanol increases calcium permeability of heavy sarcoplasmic reticulum of skeletal muscle

The effects of ethanol on both Ca2+ pump activity and Ca2+-induced Ca2+ release in sarcoplasmic reticulum (SR) of rabbit skeletal muscle were studied. In concentrations of 0.1-1.0%, ethanol conspicuously enhanced Ca2+ release from the heavy fraction of SR, whereas a much smaller effect was noted in the light fraction. When Ca2+-induced Ca2+ release was inhibited by 10 mM Mg2+, the Ca2+ pump activities of both the heavy and light SR were the same; the activities were not significantly influenced by 1% ethanol. Ethanol itself did not release Ca2+ from the heavy SR. However, it appeared to render the heavy SR more permeable to Ca2+, thereby decreasing the amount of storable Ca2+. This action of ethanol may be related to the mechanism of its negative inotropic effect.