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.     

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.     

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.     

Analysis of excitation-contraction-coupling components in chronically stimulated canine skeletal muscle.

The chronic stimulation of predominantly fast-twitch mammalian skeletal muscle causes a transformation to physiological characteristics of slow-twitch skeletal muscle. Here, we report the effects of chronic stimulation on the protein components of the sarcoplasmic reticulum and transverse tubular membranes which are directly involved in excitation-contraction coupling. Comparison of protein composition of microsomal fractions from control and chronically stimulated muscle was performed by immunoblot analysis and also by staining with Coomassie blue or the cationic carbocyanine dye Stains-all. Consistent with previous experiments, a greatly reduced density was observed for the fast-twitch isozyme of Ca(2+)-ATPase, while the expression of the slow-twitch Ca(2+)-ATPase was found to be greatly enhanced. Components of the sarcolemma (Na+/K(+)-ATPase, dystrophin-glycoprotein complex) and the free sarcoplasmic reticulum (Ca(2+)-binding protein sarcalumenin and a 53-kDa glycoprotein) were not affected by chronic stimulation. The relative abundance of calsequestrin was slightly reduced in transformed skeletal muscle. However, the expression of the ryanodine receptor/Ca(Ca2+)-release channel from junctional sarcoplasmic reticulum and the transverse tubular dihydropyridine-sensitive Ca2+ channel, as well as two junctional sarcoplasmic reticulum proteins of 90 kDa and 94 kDa, was greatly suppressed in transformed muscle. Thus, the expression of the major protein components of the triad junction involved in excitation-contraction coupling is suppressed, while the expression of other muscle membrane proteins is not affected in chronically stimulated muscle.     

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.     

Ca2+ signaling in microdomains: Homer1 mediates the interaction between RyR2 and Cav1.2 to regulate excitation-contraction coupling

Excitation-contraction (E-C) coupling and Ca(2+)-induced Ca(2+) release in smooth and cardiac muscles is mediated by the L-type Ca(2+) channel isoform Ca(v)1.2 and the ryanodine receptor isoform RyR2. Although physical coupling between Ca(v)1.1 and RyR1 in skeletal muscle is well established, it is generally assumed that Ca(v)1.2 and RyR2 do not directly communicate either passively or dynamically during E-C coupling. In the present work, we re-examined this assumption by studying E-C coupling in the detrusor muscle of wild type and Homer1(-/-) mice and by demonstrating a Homer1-mediated dynamic interaction between Ca(v)1.2 and RyR2 using the split green fluorescent protein technique. Deletion of Homer1 in mice (but not of Homer2 or Homer3) resulted in impaired urinary bladder function, which was associated with higher sensitivity of the detrusor muscle to muscarinic stimulation and membrane depolarization. This was not due to an altered expression or function of RyR2 and Ca(v)1.2. Most notably, expression of Ca(v)1.2 and RyR2 tagged with the complementary C- and N-terminal halves of green fluorescent protein and in the presence and absence of Homer1 isoforms revealed that H1a and H1b/c reciprocally modulates a dynamic interaction between Ca(v)1.2 and RyR2 to regulate the intensity of Ca(2+)-induced Ca(2+) release and its dependence on membrane depolarization. These findings define the molecular basis of a "two-state" model of E-C coupling by Ca(v)1.2 and RyR2. In one state, Ca(v)1.2 couples to RyR2 by H1b/c, which results in reduced responsiveness to membrane depolarization and in the other state H1a uncouples Ca(v)1.2 and RyR2 to enhance responsiveness to membrane depolarization. These findings reveal an unexpected and novel mode of interaction and communication between Ca(v)1.2 and RyR2 with important implications for the regulation of smooth and possibly cardiac muscle E-C coupling.     

Calsequestrin binds to monomeric and complexed forms of key calcium-handling proteins in native sarcoplasmic reticulum membranes from rabbit skeletal muscle.

Ca(2+)-handling proteins are important regulators of the excitation-contraction-relaxation cycle in skeletal muscle fibres. Although domain binding studies suggest protein coupling between various Ca(2+)-regulatory elements of triad junctions, no direct biochemical evidence exists demonstrating high-molecular-mass complex formation in native microsomal membranes. Calsequestrin represents the protein backbone of the luminal Ca(2+) reservoir and thereby occupies a central position in Ca(2+) homeostasis; we therefore used calsequestrin blot overlay assays in order to determine complex formation between sarcoplasmic reticulum components. Peroxidase-conjugated calsequestrin clearly labelled four major protein bands in one-dimensional (1D) and 2D electrophoretically separated membrane preparations from adult skeletal muscle. Immunoblotting identified the calsequestrin-binding proteins of approximately 26, 63, 94 and 560 kDa as junctin, calsequestrin itself, triadin and the ryanodine receptor, respectively. Protein-protein coupling could be modified by ionic detergents, non-ionic detergents, changes in Ca(2+) concentration, as well as antibody and purified calsequestrin binding. Importantly, complex formation as determined by blot overlay assays was confirmed by differential co-immunoprecipitation experiments and chemical crosslinking analysis. Hence, the key Ca(2+)-regulatory membrane components of skeletal muscle form a supramolecular membrane assembly. The formation of this tightly associated junctional sarcoplasmic reticulum complex seems to underlie the physiological regulation of skeletal muscle contraction and relaxation, which supports the biochemical concept that Ca(2+) homeostasis is regulated by direct protein-protein interactions.     

Co-expression of MG29 and ryanodine receptor leads to apoptotic cell death: effect mediated by intracellular Ca2+ release

Perturbation of intracellular Ca2+ homeostasis has been shown to regulate the process of cell proliferation and apoptosis. Our previous studies show that mitsugumin 29 (MG29), a synaptophysin-related protein localized in the triad junction of skeletal muscle, serves an essential role in muscle Ca2+ signaling by regulating the process of store-operated Ca2+ entry. Here we report a functional interaction between MG29 and the ryanodine receptor (RyR)/Ca2+ release channel. The purified MG29 protein enhances activity of the RyR/Ca2+ release channel incorporated into the lipid bilayer membrane. Co-expression of MG29 and RyR in Chinese hamster ovary cells leads to apoptotic cell death resulting from depletion of intracellular Ca2+ stores, despite neither protein expression alone exhibits any significant effect on cell viability. In transient expression studies, the presence of RyR in the endoplasmic reticulum leads to retention of MG29 from the plasma membrane into the intracellular organelles. This functional interaction between MG29 and RyR could have important implications in the Ca2+ signaling processes of muscle cells. Our data also show that perturbation of intracellular Ca2+ homeostasis can serve as a key signal in the initiation of apoptosis.     

Defective maintenance of intracellular Ca^2＋ homeostasis is linked to increasedmuscle fatigability in the MG29 null mice

Mitsugumin 29 (MG29) is a transmembrane protein that is normally found in the triad junction of skeletal muscle. Our previous studies have shown that targeted deletion of rag29 from the skeletal muscle resulted in abnormality of the triad junction structure, and also increased susceptibility to muscle fatigue. To elucidate the basis of these effects, we investigated the properties of Ca^2 uptake and -release in toxin-skinned Extensor Digitorium Longus (EDL) muscle fibers from control and rag29 knockout mice. Compared with the control muscle, submaximal Ca^2 uptake into the sarcoplasmic reticulum (SR) was slower and the storage of Ca^2 inside the SR was less in the mutant muscle, due to increased leakage process of Ca^2 movement across the SR. The leakage pathway is associated with the increased sensitivity of Ca^2 /caffeine -induced Ca^2 release to myoplasmic Ca^2 . Therefore, the increased fatigability of mutant EDL muscles can result from a combination of a slowing of Ca^2 uptake, modification of Ca^2 -induced Ca^2 release (CICR), and a reduction in total SR Ca^2 content.     

Dynamic regulation of sarcoplasmic reticulum Ca(2+) content and release by luminal Ca(2+)-sensitive leak in rat ventricular myocytes.

In cardiac muscle, excitation-contraction (E-C) coupling is determined by the ability of the sarcoplasmic reticulum (SR) to store and release Ca(2+). It has been hypothesized that the Ca(2+) sequestration and release mechanisms might be functionally linked to optimize the E-C coupling process. To explore the relationships between the loading status of the SR and functional state of the Ca(2+) release mechanism, we examined the effects of changes in SR Ca(2+) content on spontaneous Ca(2+) sparks in saponin-permeabilized and patch-clamped rat ventricular myocytes. SR Ca(2+) content was manipulated by pharmacologically altering the capacities of either Ca(2+) uptake or leak. Ca(2+) sparks were recorded using a confocal microscope and Fluo-3 and were quantified considering missed events. SR Ca(2+) content was assessed by application of caffeine. Exposure of permeabilized cells to anti-phospholamban antibodies elevated the SR Ca(2+) content and increased the frequency of sparks. Suppression of the SR Ca(2+) pump by thapsigargin lowered [Ca(2+)](SR) and reduced the frequency of sparks. The ryanodine receptor (RyR) blockers tetracaine and Mg(2+) transiently suppressed the frequency of sparks. Upon washout of the drugs, sparking activity transiently overshot control levels. Low doses of caffeine transiently potentiated sparking activity upon application and transiently depressed the sparks upon removal. In patch-clamped cardiac myocytes, exposure to caffeine produced only a transient increase in the probability of sparks induced by depolarization. We interpret these results in terms of a novel dynamic control scheme for SR Ca(2+) cycling. A central element of this scheme is a luminal Ca(2+) sensor that links the functional activity of RyRs to the loading state of the SR, allowing cells to auto-regulate the size and functional state of their SR Ca(2+) pool. These results are important for understanding the regulation of intracellular Ca(2+) release and contractility in cardiac muscle.     

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.     

Structural analysis of muscle development: transverse tubules, sarcoplasmic reticulum, and the triad.

Increased interest in the mechanism of excitation-contraction (E-C) coupling over the last few years has been accompanied by numerous investigations into the development of the underlying cellular structures. Areas of particular interest include: (1) the compartmentalization and specialization of an external and an internal membrane system, the T-tubules, and the sarcoplasmic reticulum, respectively; (2) interactions between the membrane proteins of both systems upon the formation of a junction, the triad; and (3) membrane-cytoskeletal interactions leading to the orderly arrangement of the triads with respect to the myofibrils. Structural studies using newly available specific molecular probes and a variety of in vivo and in vitro model systems have provided new insights into the cellular and molecular mechanisms involved in the development of the E-C coupling apparatus in skeletal muscle.     

Triadins modulate intracellular Ca(2+) homeostasis but are not essential for excitation-contraction coupling in skeletal muscle

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca(2+) imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca(2+) transients. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca(2+).[(3)H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca(2+) activation or Ca(2+) and Mg(2+) inhibition between wild-type and triadin-null animals. Subtle ultrastructural changes, evidenced by the appearance of longitudinally oriented triads and the presence of calsequestrin in the sacs of the longitudinal SR, were present in fast but not slow twitch-null muscles. Overall, our data support an indirect role for triadin in regulating myoplasmic Ca(2+) homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling.     

Mitochondrial calcium uptake regulates rapid calcium transients in skeletal muscle during excitation-contraction (E-C) coupling

Defective coupling between sarcoplasmic reticulum and mitochondria during control of intracellular Ca(2+) signaling has been implicated in the progression of neuromuscular diseases. Our previous study showed that skeletal muscles derived from an amyotrophic lateral sclerosis (ALS) mouse model displayed segmental loss of mitochondrial function that was coupled with elevated and uncontrolled sarcoplasmic reticulum Ca(2+) release activity. The localized mitochondrial defect in the ALS muscle allows for examination of the mitochondrial contribution to Ca(2+) removal during excitation-contraction coupling by comparing Ca(2+) transients in regions with normal and defective mitochondria in the same muscle fiber. Here we show that Ca(2+) transients elicited by membrane depolarization in fiber segments with defective mitochondria display an ~10% increased amplitude. These regional differences in Ca(2+) transients were abolished by the application of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a fast Ca(2+) chelator that reduces mitochondrial Ca(2+) uptake. Using a mitochondria-targeted Ca(2+) biosensor (mt11-YC3.6) expressed in ALS muscle fibers, we monitored the dynamic change of mitochondrial Ca(2+) levels during voltage-induced Ca(2+) release and detected a reduced Ca(2+) uptake by mitochondria in the fiber segment with defective mitochondria, which mirrored the elevated Ca(2+) transients in the cytosol. Our study constitutes a direct demonstration of the importance of mitochondria in shaping the cytosolic Ca(2+) signaling in skeletal muscle during excitation-contraction coupling and establishes that malfunction of this mechanism may contribute to neuromuscular degeneration in ALS.     

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.     

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)     

兔膈肌骨骼肌型DHPRα1亚单位和RyRs的mRNA与蛋白表达测定

本文测定了兔膈肌钙释放单位中骨骼肌型ＤＨＰＲα１－亚单位和ＲｙＲｓ的ｍＲＮＡ与蛋白表达水平,探讨了兔膈肌钙释放单位的结构组成特征。采用ＲＴ－ＰＣＲ、原位杂交和免疫组织化学技术,分别测定兔膈肌骨骼肌型ＤＨＰＲα１－亚单位和ＲｙＲ１、ＲｙＲ２及ＲｙＲ３的ｍＲＮＡ与蛋白表达。结果显示,兔膈肌可见较高水平的骨骼肌型α１－亚单位和ＲｙＲ１ｍＲＮＡ与蛋白表达;较低水平的ＲｙＲ３ ｍＲＮＡ与蛋白表达。表明兔膈肌     

Specific absence of the alpha 1 subunit of the dihydropyridine receptor in mice with muscular dysgenesis

Muscular dysgenesis is a lethal mutation in mice that results in a complete absence of skeletal muscle contraction due to the failure of depolarization of the transverse tubular membrane to trigger calcium release from the sarcoplasmic reticulum. In order to determine whether the defect in muscular dysgenesis leads to a specific loss of one of the components of excitation-contraction coupling or to a generalized loss of all components of excitation-contraction coupling, we have analyzed skeletal muscle from control and dysgenic mice for the sarcoplasmic reticulum and transverse tubular proteins which are believe to function in excitation-contraction coupling. We report that the proteins involved in sarcoplasmic reticulum calcium transport, storage, and release [Ca2+ + Mg2+)-ATPase, calsequestrin, and calcium release channel) are present in dysgenic muscle. Also present in dysgenic muscle is the 175/150-kDa glycoprotein subunit (alpha 2) of the dihydropyridine receptor. However, the 170-kDa dihydropyridine binding subunit (alpha 1) of the dihydropyridine receptor is absent in dysgenic muscle. These results suggest that the specific absence of the alpha 1 subunit of the dihydropyridine receptor is responsible for the defects in muscular dysgenesis and that the alpha 1 subunit of the dihydropyridine receptor is essential for excitation-contraction coupling in skeletal muscle.