Abnormal features in skeletal muscle from mice lacking mitsugumin29

Physiological roles of the members of the synaptophysin family, carrying four transmembrane segments and being basically distributed on intracellular membranes including synaptic vesicles, have not been established yet. Recently, mitsugumin29 (MG29) was identified as a novel member of the synaptophysin family from skeletal muscle. MG29 is expressed in the junctional membrane complex between the cell surface transverse (T) tubule and the sarcoplasmic reticulum (SR), called the triad junction, where the depolarization signal is converted to Ca(2+) release from the SR. In this study, we examined biological functions of MG29 by generating knockout mice. The MG29-deficient mice exhibited normal health and reproduction but were slightly reduced in body weight. Ultrastructural abnormalities of the membranes around the triad junction were detected in skeletal muscle from the mutant mice, i.e., swollen T tubules, irregular SR structures, and partial misformation of triad junctions. In the mutant muscle, apparently normal tetanus tension was observed, whereas twitch tension was significantly reduced. Moreover, the mutant muscle showed faster decrease of twitch tension under Ca(2+)-free conditions. The morphological and functional abnormalities of the mutant muscle seem to be related to each other and indicate that MG29 is essential for both refinement of the membrane structures and effective excitation-contraction coupling in the skeletal muscle triad junction. Our results further imply a role of MG29 as a synaptophysin family member in the accurate formation of junctional complexes between the cell surface and intracellular membranes.     

Junctophilins: a novel family of junctional membrane complex proteins

Junctional complexes between the plasma membrane (PM) and endoplasmic/sarcoplasmic reticulum (ER/ SR) are a common feature of all excitable cell types and mediate cross-talk between cell surface and intracellular ion channels. We have identified the junctophilins (JPs), a novel conserved family of proteins that are components of the junctional complexes. JPs are composed of a carboxy-terminal hydrophobic segment spanning the ER/SR membrane and a remaining cytoplasmic domain that shows specific affinity for the PM. In mouse, there are at least three JP subtypes: JP-1, -2, and -3. JP-2 is abundantly expressed in the heart, and mutant mice lacking JP-2 exhibited embryonic lethality. Cardiac myocytes from the mutant mice showed deficiency of the junctional membrane complexes and abnormal Ca2+ transients. Our results suggest that JPs are important components of junctional membrane complexes.     

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.     

Embryonic lethality and abnormal cardiac myocytes in mice lacking ryanodine receptor type 2.

The ryanodine receptor type 2 (RyR-2) functions as a Ca2+-induced Ca2+ release (CICR) channel on intracellular Ca2+ stores and is distributed in most excitable cells with the exception of skeletal muscle cells. RyR-2 is abundantly expressed in cardiac muscle cells and is thought to mediate Ca2+ release triggered by Ca2+ influx through the voltage-gated Ca2+ channel to constitute the cardiac type of excitation-contraction (E-C) coupling. Here we report on mutant mice lacking RyR-2. The mutant mice died at approximately embryonic day (E) 10 with morphological abnormalities in the heart tube. Prior to embryonic death, large vacuolate sarcoplasmic reticulum (SR) and structurally abnormal mitochondria began to develop in the mutant cardiac myocytes, and the vacuolate SR appeared to contain high concentrations of Ca2+. Fluorometric Ca2+ measurements showed that a Ca2+ transient evoked by caffeine, an activator of RyRs, was abolished in the mutant cardiac myocytes. However, both mutant and control hearts showed spontaneous rhythmic contractions at E9.5. Moreover, treatment with ryanodine, which locks RyR channels in their open state, did not exert a major effect on spontaneous Ca2+ transients in control cardiac myocytes at E9.5-11.5. These results suggest no essential contribution of the RyR-2 to E-C coupling in cardiac myocytes during early embryonic stages. Our results from the mutant mice indicate that the major role of RyR-2 is not in E-C coupling as the CICR channel in embryonic cardiac myocytes but it is absolutely required for cellular Ca2+ homeostasis most probably as a major Ca2+ leak channel to maintain the developing SR.     

Store-operated Ca2+ entry uncoupled with ryanodine receptor and junctional membrane complex in heart muscle cells

Store-operated Ca2+ entry (SOCE) is the Ca2+ influx that is activated on depletion of intracellular Ca2+ stores. Although SOCE is found in a variety of cell types, its activation mechanism and molecular identity remain to be clarified. Current experimental results suggest that SOCE channels are activated by direct coupling with Ca2+ release channels on depleted stores. Here we report SOCE in cardiac myocytes, that was prominently sensitive to Zn2+ but resistant to inhibitors for voltage-dependent Ca2+ channels and Na+/Ca2+ exchangers. The SOCE activity may be developmentally regulated, because the SOCE was easily detected during embryonic and neonatal stages but not in mature myocytes from adult hearts. In cardiac myocytes, ryanodine receptor type 2 (RyR-2) is thought to be the sole Ca2+ release channel on the intracellular store, and junctophilin type 2 (JP-2) contributes to formation of the junctional complex between the cell surface and store membranes. Using the knockout mice, we also examined possible involvement of the Ca2+ release channel and junctional membrane complex in cardiac SOCE. Apparently normal SOCE activities were retained in mutant myocytes lacking RyR-2 or JP-2, suggesting that neither the Ca2+ release channel nor junctional membrane complex is involved in activation of cardiac SOCE.     

Calyculin-A disrupts subplasmalemmal junction and recurring Ca2+ waves in vascular smooth muscle

Excitation-contraction coupling (E-C coupling) in phenylephrine(PE)-stimulated rabbit inferior vena cava (IVC) depends on the generation of asynchronous recurring Ca2+ waves in the in situ vascular smooth muscle cells (VSMC). Previous studies by our group have implicated a putative non-selective cationic store-operated channel and the reverse-mode Na+-Ca2+ exchange in refilling of the intracellular Ca2+ store via the sarco/endoplasmic reticulum ATPase (SERCA) and the maintenance of the recurring Ca2+ waves. We hypothesize that for the proper functioning of these three Ca2+ translocators in the process of SR refilling, the plasma membrane (PM) and the underlying superficial sarcoplasmic reticulum (SR) form specialized PM-SR junctions, which are essential for the maintenance of the recurring Ca2+ waves. In order to test this hypothesis, calyculin-A, a serine/threonine phosphatase inhibitor that has been demonstrated to result in the disruption of the PM-SR junctions was used. In the control rabbit IVC, electron microscopy of the in situ VSMC indicates that 14.2+/-0.7% of the PM is closely apposed by the prominent superficial SR network, forming numerous flattened PM-SR junctional cytoplasmic spaces. In the control IVC stimulation with 5 microM PE resulted in sustained recurring Ca2+ waves with a frequency of 0.42+/-0.02 Hz. In calyculin-A treated rabbit IVC, a concentration-dependent dissociation of the superficial SR and loss of PM-SR junctions was observed. This progressive loss of the PM-SR junctions occurs over the same concentration range as the inhibition of PE-induced recurring Ca2+ waves. These findings offer support for the hypothesis that the presence of the PM-SR junctions is required for the generation of asynchronous recurring Ca2+ waves, which underlie excitation-contraction coupling in the VSMC of the rabbit IVC.     

Deficiency of triad formation in developing skeletal muscle cells lacking junctophilin type 1

Junctophilins (JP-1, JP-2, and JP-3) are transmembrane proteins expressed in the junctional membrane complexes in excitable cells. Both JP-1 and JP-2 are co-expressed in the triads of skeletal muscle, but only JP-2 is expressed in cardiac muscle. We analyzed the roles played by JP-1 and JP-2 in triad formation in skeletal muscle by comparing developing skeletal muscles in wild-type and JP-1-knockout (KO) mice (both before and after birth). In the skeletal muscles of embryos, most of the couplings between sarcoplasmic reticulum (SR) and transverse tubule (T-tubule) were diads, with triads being very scarce. The number of triads increased markedly after birth in wild-type mice. However, there was no increase in the number of triads in the neonates of JP-1-KO mice, and they died within 1 day after birth. JP-2 expression was constant before and after birth, while expression of JP-1 increased with birth. Quantitative and morphological differences were not seen between wild-type and JP-1-KO mice in the formation of diads in the period just before the JP-1-KO mice died. The SR swelled and developed large vacuoles in skeletal muscle cells just before the JP-1-KO mice died. The present results strongly suggest that JP-1 and JP-2 play important roles in the formation of triads and diads, respectively, during the development of skeletal muscle in mouse.     

Molecular organization of transverse tubule/sarcoplasmic reticulum junctions during development of excitation-contraction coupling in skeletal muscle.

The relationship between the molecular composition and organization of the triad junction and the development of excitation-contraction (E-C) coupling was investigated in cultured skeletal muscle. Action potential-induced calcium transients develop concomitantly with the first expression of the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR), which are colocalized in clusters from the time of their earliest appearance. These DHPR/RyR clusters correspond to junctional domains of the transverse tubules (T-tubules) and sarcoplasmic reticulum (SR), respectively. Thus, at first contact T-tubules and SR form molecularly and structurally specialized membrane domains that support E-C coupling. The earliest T-tubule/SR junctions show structural characteristics of mature triads but are diverse in conformation and typically are formed before the extensive development of myofibrils. Whereas the initial formation of T-tubule/SR junctions is independent of association with myofibrils, the reorganization into proper triads occurs as junctions become associated with the border between the A band and the I band of the sarcomere. This final step in triad formation manifests itself in an increased density and uniformity of junctions in the cytoplasm, which in turn results in increased calcium release and reuptake rates.     

Mechanisms of excitation-contraction coupling and calcium liberation in striated muscles of vertebrates

In vertebrate skeletal muscle, the main part of excitation-contraction coupling occurs at the level of the triad, where membranes of T-system and of junctional SR are facing each other. From place to place, the junctional gap is bridged by "feet" structures which include the SR Ca2+ channel. Half of them are closely apposed to tubular intramembranous structures assumed to be DHP-sensitive voltage-sensors which are similar to tubular Ca2+ channels and act by controlling Ca2+ release from SR. During a twitch, the release of Ca2+ activator from SR is controlled both by voltage-sensors via the feet structures and by a tubular Na+ current via a Na+-induced Ca2+ release mechanism. During long-duration mechanical responses, additional mechanisms are involved: a Ca2+-induced Ca2+ release which can be activated by ICa; the release of Ca2+ from membrane, controlled by the operation of a Na+/Ca2+ exchanger and/or new arrangements of surface membrane charges. An IP3-mediated Ca2+ release could be involved too. All these mechanisms can be regulated by intracellular biochemical or ionic processes.     

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.     

Formation of triads without the dihydropyridine receptor alpha subunits in cell lines from dysgenic skeletal muscle

Muscular dysgenesis (mdg/mdg), a mutation of the skeletal muscle dihydropyridine receptor (DHPR) alpha 1 subunit, has served as a model to study the functions of the DHPR in excitation-contraction coupling and its role in triad formation. We have investigated the question of whether the lack of the DHPR in dysgenic skeletal muscle results in a failure of triad formation, using cell lines (GLT and NLT) derived from dysgenic (mdg/mdg) and normal (+/+) muscle, respectively. The lines were generated by transfection of myoblasts with a plasmid encoding a Large T antigen. Both cell lines express muscle-specific proteins and begin organization of sarcomeres as demonstrated by immunocytochemistry. Similar to primary cultures, dysgenic (GLT) myoblasts show a higher incidence of cell fusion than their normal counterparts (NLT). NLT myotubes develop spontaneous contractile activity, and fluorescent Ca2+ recordings show Ca2+ release in response to depolarization. In contrast, GLTs show neither spontaneous nor depolarization-induced Ca2+ transients, but do release Ca2+ from the sarcoplasmic reticulum (SR) in response to caffeine. Despite normal transverse tubule (T-tubule) formation, GLT myotubes lack the alpha 1 subunit of the skeletal muscle DHPR, and the alpha 2 subunit is mistargeted. Nevertheless, the ryanodine receptor (RyR) frequently develops its normal, clustered organization in the absence of both DHPR alpha subunits in the T-tubules. In EM, these RyR clusters correspond to T-tubule/SR junctions with regularly spaced feet. These findings provide conclusive evidence that interactions between the DHPR and RyR are not involved in the formation of triad junctions or in the normal organization of the RyR in the junctional SR.     

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.     

Dihydropyridine receptor-ryanodine receptor interactions in skeletal muscle excitation-contraction coupling

Much recent progress has been made in our understanding of the mechanism of sarcoplasmic reticulum Ca²⁺ release in skeletal muscle. Vertebrate skeletal muscle excitation-contraction (E-C) coupling is thought to occur by a mechanical coupling mechanism involving protein-protein interactions that lead to activation of the sarcoplasmic reticulum (SR) ryanodine receptor (RyR)/Ca²⁺ release channel by the voltage-sensing transverse (T–) tubule dihydropyridine receptor (DHPR)/Ca²⁺ channel. In a subsequent step, the released Ca²⁺ amplify SR Ca²⁺ release by activating release channels that are not linked to the DHPR. Experiments with mutant muscle cells have indicated that skeletal muscle specific DHPR and RyR isoforms are required for skeletal muscle E-C coupling. A direct functional and structural interaction between a DHPR-derived peptide and the RyR has been described. The interaction between the DHPR and RyR may be stabilized by other proteins such as triadin (a SR junctional protein) and modulated by phosphorylation of the DHPR.     

Immuno-proteomic approach to excitation--contraction coupling in skeletal and cardiac muscle: molecular insights revealed by the mitsugumins

A comprehensive understanding of excitation-contraction (E-C) coupling in skeletal and cardiac muscle requires that all the major components of the Ca(2+) release machinery be resolved. We utilized a unique immuno-proteomic approach to generate a monoclonal antibody library that targets proteins localized to the skeletal muscle triad junction, which provides a structural context to allow efficient E-C coupling. Screening of this library has identified several mitsugumins (MG); proteins that can be localized to the triad junction in mammalian skeletal muscle. Many of these proteins, including MG29 and junctophilin, are important components in maintaining the structural integrity of the triad junction. Other triad proteins, such as calumin, play a more direct role in regulation of muscle Ca(2+) homeostasis. We have recently identified a family of trimeric intracellular cation-selective (TRIC) channels that allow for K(+) movement into the endoplasmic or sarcoplasmic reticulum to counter a portion of the transient negative charge produced by Ca(2+) release into the cytosol. Further study of TRIC channel function and other novel mitsugumins will increase our understanding of E-C coupling and Ca(2+) homoeostasis in muscle physiology and pathophysiology.     

Subtype specificity of the ryanodine receptor for Ca2+ signal amplification in excitation-contraction coupling.

In excitable cells membrane depolarization is translated into intracellular Ca2+ signals. The ryanodine receptor (RyR) amplifies the Ca2+ signal by releasing Ca2+ from the intracellular Ca2+ store upon receipt of a message from the dihydropyridine receptor (DHPR) on the plasma membrane in striated muscle. There are two distinct mechanisms for the amplification of Ca2+ signalling. In cardiac cells depolarization-dependent Ca2+ influx through DHPR triggers Ca2+-induced Ca2+ release via RyR, while in skeletal muscle cells a voltage-induced change in DHPR is thought to be mechanically transmitted, without a requirement for Ca2+ influx, to RyR to cause it to open. In expression experiments using mutant skeletal myocytes lacking an intrinsic subtype of RyR (RyR-1), we demonstrate that RyR-1, but not the cardiac subtype (RyR-2), is capable of supporting skeletal muscle-type coupling. Furthermore, when RyR-2 was expressed in skeletal myocytes, we observed depolarization-independent spontaneous Ca2+ waves and oscillations, which suggests that RyR-2 is prone to regenerative Ca2+ release responses. These results demonstrate functional diversity among RyR subtypes and indicate that the subtype of RyR is the key to Ca2+ signal amplification.     

Pharmacological studies of excitation-contraction coupling in skeletal muscle

The use of drugs in the study of excitation-contraction (E-C) coupling in skeletal muscle during the 25-30 years and the role of these studies in the development of the "trigger-calcium" hypothesis was reviewed. In early studies, caffeine was used as a tool to test the function of the intracellular contraction apparatus when the twitch or depolarization contracture was eliminated by a procedure that was thought to block the coupling part of the E-C coupling process. Later it was shown that caffeine produced contractures by releasing Ca2+ ions from intracellular binding sites and then that caffeine produced this effect by sensitizing the sarcoplasmic reticulum to Ca2+-induced Ca2+ release. More recently, organic calcium channel blocking drugs (verapamil, D-600, and nitrendipine) were used to confirm earlier results showing that depolarization contractures but not twitches require the entrance into the cells via the slow Ca2+ channels of extracellular calcium ions for E-C coupling. Most recently, we have investigated the effects of TMB-8 (8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate) on E-C coupling in frog skeletal muscle. This compound was shown by other workers to act in several tissues by stabilizing Ca2+ bound at intracellular sites. It was found that at the appropriate concentration TMB-8 blocked twitches but neither high K+ nor caffeine induced contractures. These results suggest that TMB-8 blocks twitches by preventing the release of Ca2+ ions bound to the intracellular surface of the t-tubular membrane, which is often called the store of "trigger-calcium" ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage clamp methods for the study of membrane currents and SR Ca(2+) release in adult skeletal muscle fibres

Skeletal muscle excitation-contraction (E-C)(1) coupling is a process composed of multiple sequential stages, by which an action potential triggers sarcoplasmic reticulum (SR)(2) Ca(2+) release and subsequent contractile activation. The various steps in the E-C coupling process in skeletal muscle can be studied using different techniques. The simultaneous recordings of sarcolemmal electrical signals and the accompanying elevation in myoplasmic Ca(2+), due to depolarization-initiated SR Ca(2+) release in skeletal muscle fibres, have been useful to obtain a better understanding of muscle function. In studying the origin and mechanism of voltage dependency of E-C coupling a variety of different techniques have been used to control the voltage in adult skeletal fibres. Pioneering work in muscles isolated from amphibians or crustaceans used microelectrodes or 'high resistance gap' techniques to manipulate the voltage in the muscle fibres. The development of the patch clamp technique and its variant, the whole-cell clamp configuration that facilitates the manipulation of the intracellular environment, allowed the use of the voltage clamp techniques in different cell types, including skeletal muscle fibres. The aim of this article is to present an historical perspective of the voltage clamp methods used to study skeletal muscle E-C coupling as well as to describe the current status of using the whole-cell patch clamp technique in studies in which the electrical and Ca(2+) signalling properties of mouse skeletal muscle membranes are being investigated.     

Characterization of human junctophilin subtype genes

Junctophilin (JP) subtypes, namely JP-1, 2, and 3, have been currently identified in excitable cells and constitute a novel family of junctional membrane complex proteins. Our studies have suggested that JPs take part in the formation of junctional membrane complexes by spanning the membrane of the intracellular Ca(2+) store and interacting with the cell-surface membrane. In this report we describe the primary structures, genomic organization, and tissue distribution of human JP subtypes. By cloning and analyzing human genomic DNA segments, the protein-coding sequence interrupted with four introns was defined in each JP gene. The deduced human JP subtypes shared characteristic structural features with their rabbit and mouse counterparts. Genomic mapping demonstrated that JP genes do not cluster on the human genome. RNA blot hybridization indicated that tissue-specific expression patterns of JP genes in human are essentially the same as those in mouse; skeletal muscle contained both JP-1 and JP-2 mRNAs, the heart predominantly expressed JP-2 mRNA, and the brain specifically contained JP-3 mRNA. In the light of this, we propose intramolecular domains of JP subtypes based on the structural and functional characteristics.     

The 90-kDa junctional sarcoplasmic reticulum protein forms an integral part of a supramolecular triad complex in skeletal muscle.

Although it is well established that voltage-sensing of the alpha(1)-dihydropyridine receptor triggers Ca(2+)-release via the ryanodine receptor during excitation-contraction coupling in skeletal muscle fibers, it remains to be determined which junctional components are responsible for the assembly, maintenance, and stabilization of triads. Here, we analyzed the expression pattern and neighborhood relationship of a novel 90-kDa sarcoplasmic reticulum protein. This protein is highly enriched in the triad fraction and is predominantly expressed in fast-twitching muscle fibers. Chronic low-frequency electro-stimulation induced a drastic decrease in the relative abundance of this protein. Chemical crosslinking showed a potential overlap between the 90-kDa junctional face membrane protein and the ryanodine receptor Ca(2+)-release channel, suggesting tight protein-protein interactions between these two triad components. Hence, Ca(2+)-regulatory muscle proteins have a strong tendency to oligomerize and the triad region of skeletal muscle fibers forms supramolecular membrane complexes involved in the regulation of Ca(2+)-homeostasis and signal transduction.     

Functional studies of Ca2+ channels from plasmalemma and sarcoplasmic reticulum membranes in muscle cells

Physiological and biochemical studies (channel characteristics, intracellular Ca2+ determinations and, channel purification, cloning and expression) of the different components involved in the regulation of intercellular Ca2+ have provided new information about their specific role. Recent information favors a major role for plasmalemma Ca2+ channels in E-C coupling of cardiac muscle, while a major role for sarcoplasmic reticulum Ca2+ release channels (ryanodine receptors) is proposed for E-C coupling of skeletal muscle. In smooth muscle, both plasmalemma and sarcoplasmic reticulum (IP3 receptors) Ca2+ channels are involved in E-C coupling. These studies will be comparatively discussed for skeletal, cardiac and smooth muscle cells.