Detection and localization of triadin in rat ventricular muscle

Summary Dyads (transverse tubule—junctional sarcoplasmic reticulum complexes) were enriched from rat ventricle microsomes by continuous sucrose gradients. The major vesicle peak at 36% sucrose contained up to 90% of those membranes which possessed dihydropyridine (DHP) binding sites (markers for transverse tubules) and all membranes which possessed ryanodine receptors and the putative junctional foot protein (markers for junctional sarcoplasmic reticulum). In addition, the 36% sucrose peak contained half of the vesicles with muscarine receptors. Vesicles derived from the nonjunctional plasma membrane as defined by a low content of dihydropyridine binding sites per muscarine receptor and from the free sarcoplasmic reticulum as defined by the M_r 102K Ca²⁺ ATPase were associated with a diffuse protein band (22–30% sucrose) in the lighter region of the gradient. These organelles were recovered in low yield. Putative dyads were not broken by French press treatment at 8,000 psi and only partially disrupted at 14,000 psi. The monoclonal antibody GE4.90 against skeletal muscle triadin, a protein which links the DHP receptor to the junctional foot protein in skeletal muscle triad junctions, cross-reacted with a protein in rat dyads of the same M_r as triadin. Western blots of muscle microsomes from preparations which had been treated with 100mm iodoacetamide throughout the isolation procedure showed that cardiac triadin consisted predominantly of a band of Mr 95 kD. Higher molecular weight polymers were detectable but low in content, in contrast with the ladder of oligomeric forms in rat psoas muscle microsomes. Cardiac triadin was not dissolved from the microsomes by hypertonic salt or Triton X-100, indicating that it, as well as skeletal muscle triadin, was an integral protein of the junctional SR. The cardiac epitope was localized to the junctional SR by comparison of its distribution with that of organelle markers in both total microsome and in French press disrupted dyad preparations. Immunofluorescence localization of triadin using mAb GE4.90 revealed that intact rat ventricular muscle tissue was stained following a well-defined pattern of bands every sarcomere. This spacing of bands was consistent with the interpretation that triadin was present in the dyadic junctional regions.     

Identification of two populations of cardiac microsomes with nitrendipine receptors: correlation of the distribution of dihydropyridine receptors with organelle specific markers

Conventional sarcolemma and microsome preparations from rabbit and cat ventricular muscle were fractionated on continuous linear sucrose gradients. The distribution of nitrendipine receptors was compared with the distribution of organelle specific markers. For the conventional sarcolemma preparation, the dihydropyridine receptor distribution matched the pattern for external membrane markers in position and shape. The number of nitrendipine receptors was three times the number of muscarine binding sites (approximately 1.0 pmol/mg protein) at the isopycnic point of the vesicles. In contrast, two populations of vesicles with nitrendipine receptors were found in the microsome preparations. One population banded with the external membrane vesicles at a mean buoyant density of 24% (w/w) sucrose. The specific content of dihydropyridine receptors (0.2 pmol/mg) was 1/5 that for the muscarine receptors. The second and major population followed the distribution of an Mr 300K polypeptide, a marker for the junctional cisternae of the sarcoplasmic reticulum (SR). Muscarine receptors, however, were also present throughout that band, albeit at a reduced specific content (approximately 0.1 pmol/mg) compared to the light vesicles. The nitrendipine specific content increased over threefold from that of the light vesicles such that the relative content (nitrendipine/muscarine) was twice that determined for the conventional sarcolemma preparation. Nitrendipine receptors were not associated with nonjunctional SR or mitochondria. The light and heavy microsome populations were incubated with 0.2 mg digitonin/mg protein, a treatment which preferentially perturbs the isopycnic point of external membrane vesicles. For the light vesicles, the membranes with muscarine and nitrendipine receptors became heavier than the bulk of the SR. In contrast, after digitonin treatment of the heavy vesicle population, the nitrendipine and muscarine receptors and the SR marker appeared to comigrate into a sharpened band at 39% sucrose. The possibility that the dihydropyridine binding sites in the heavy microsome population are on external membrane vesicles physically linked to the junctional SR is discussed.     

Muscle fibers from dysgenic mouse in vivo lack a surface component of peripheral couplings.

We have studied the structure of developing normal and dysgenic (mdg/mdg) mouse muscle fibers in vivo, with special attention to the components of the junctions between the sarcoplasmic reticulum and either the surface membrane or the transverse tubules. Triads and dyads are rare in dysgenic muscle fibers, but have apparently normal disposition of feet and calsequestrin. Peripheral couplings in normal developing muscle fibers have junctional tetrads in their surface membrane in association with the junctional feet. Muscle fibers in dysgenic mice lack junctional tetrads. This provides indirect evidence for the identification of the components of junctional tetrads with dihydropyridine receptors, which are known to be absent in dysgenic muscle fibers.     

Molecular architecture of membranes involved in excitation-contraction coupling of cardiac muscle

Peripheral couplings are junctions between the sarcoplasmic reticulum (SR) and the surface membrane (SM). Feet occupy the SR/SM junctional gap and are identified as the SR calcium release channels, or ryanodine receptors (RyRs). In cardiac muscle, the activation of RyRs during excitation-contraction (e-c) coupling is initiated by surface membrane depolarization, followed by the opening of surface membrane calcium channels, the dihydropyridine receptors (DHPRs). We have studied the disposition of DHPRs and RyRs, and the structure of peripheral couplings in chick myocardium, a muscle that has no transverse tubules. Immunolabeling shows colocalization of RyRs and DHPRs in clusters at the fiber's periphery. The positions of DHPR and RyR clusters change coincidentally during development. Freeze-fracture of the surface membrane reveals the presence of domains (junctional domains) occupied by clusters of large particles. Junctional domains in the surface membrane and arrays of feet in the junctional gap have similar sizes and corresponding positions during development, suggesting that both are components of peripheral couplings. As opposed to skeletal muscle, membrane particles in junctional domains of cardiac muscle do not form tetrads. Thus, despite their proximity to the feet, they do not appear to be specifically associated with them. Two observations establish the identify of the structurally identified feet arrays/junctional domain complexes with the immunocytochemically defined RyRs/DHPRs coclusters: the concomitant changes during development and the identification of feet as the cytoplasmic domains of RyRs. We suggest that the large particles in junctional domains of the surface membrane represent DHPRs. These observations have two important functional consequences. First, the apposition of DHPRs and RyRs indicates that most of the inward calcium current flows into the restricted space where feet are located. Secondly, contrary to skeletal muscle, presumptive DHPRs do not show a specific association with the feet, which is consistent with a less direct role of charge movement in cardiac than in skeletal e-c coupling.     

Simultaneous maturation of transverse tubules and sarcoplasmic reticulum during muscle differentiation in the mouse

The development and maturation of transverse (T) tubules and sarcoplasmic reticulum (SR) have been studied in pre- and postnatal mouse muscle, using selective "staining" of these membrane systems. As previously reported in the literature, orderly transverse orientation of the T tubules occurs late in development and early T-SR junctions (triads and dyads) are located at random along the T tubules in a predominantly longitudinal orientation. We find that initial appearance of transverse tubules occurs fairly abruptly, and that all early T tubules have a longitudinal orientation. Transverse orientation of the T tubule network, location of triads at the A-I junction, and development of differentiated regions of the SR are coordinated events which occur gradually over a period of about 3 weeks for leg muscle.s The timing of triad development coincides with that reported for the increase in slow calcium current and dihydropyridine binding. Differences in T tubule patterns between muscle fibers of EDL and soleus are apparent only at relatively late stages.     

The Ca2+-release channel/ryanodine receptor is localized in junctional and corbular sarcoplasmic reticulum in cardiac muscle

The subcellular distribution of the Ca(2+)-release channel/ryanodine receptor in adult rat papillary myofibers has been determined by immunofluorescence and immunoelectron microscopical studies using affinity purified antibodies against the ryanodine receptor. The receptor is confined to the sarcoplasmic reticulum (SR) where it is localized to interior and peripheral junctional SR and the corbular SR, but it is absent from the network SR where the SR-Ca(2+)-ATPase and phospholamban are densely distributed. Immunofluorescence labeling of sheep Purkinje fibers show that the ryanodine receptor is confined to discrete foci while the SR-Ca(2+)-ATPase is distributed in a continuous network-like structure present at the periphery as well as throughout interior regions of these myofibers. Because Purkinje fibers lack T- tubules, these results indicate that the ryanodine receptor is localized not only to the peripheral junctional SR but also to corbular SR densely distributed in interfibrillar spaces of the I-band regions. We have previously identified both corbular SR and junctional SR in cardiac muscle as potential Ca(2+)-storage/Ca(2+)-release sites by demonstrating that the Ca2+ binding protein calsequestrin and calcium are very densely distributed in these two specialized domains of cardiac SR in situ. The results presented here provide strong evidence in support of the hypothesis that corbular SR is indeed a site of Ca(2+)-induced Ca2+ release via the ryanodine receptor during excitation contraction coupling in cardiac muscle. Furthermore, these results indicate that the function of the cardiac Ca(2+)-release channel/ryanodine receptor is not confined to junctional complexes between SR and the sarcolemma.     

FREEZE FRACTURE OF SKELETAL MUSCLE FROM THE TARANTULA SPIDER : Structural Differentiations of Sarcoplasmic Reticulum and Transverse Tubular System Membranes

The structure of the membranes of sarcoplasmic reticulum (SR), tubular (T) system, and sarcolemma has been studied by freeze fracture in leg muscles of the Tarantula spider. Two regions of the sarcoplasmic reticulum can be differentiated by the distribution of particles on the fracture faces: a junctional SR, at the dyads, and a longitudinal SR, elsewhere. The dyads are asymmetric junctions, the disposition of particles in the apposed membranes of SR and T tubules being different from one another and from the regular arrangement of feet in the junctional gap. It is concluded that no channels can be visualized to directly connect SR- and T-system lumina.     

Characterization of junctional and longitudinal sarcoplasmic reticulum from heart muscle

Longitudinal tubules and junctional sarcoplasmic reticulum (SR) were prepared from heart muscle microsomes by Ca2+-phosphate loading followed by sucrose density gradient centrifugation. The longitudinal SR had a high Ca2+ loading rate (0.93 +/- 0.08 mumol.mg-1.min) which was unchanged by addition of ruthenium red. Junctional SR had a low Ca2+ loading rate (0.16 +/- 0.02 mumol.mg-1.min) which was enhanced about 5-fold by ruthenium red. Junctional SR had feet structures observed by electron microscopy and a high molecular weight protein with Mr of 340,000, whereas longitudinal SR was essentially devoid of both. Thus, these subfractions have similar characteristics to longitudinal and junctional terminal cisternae of SR from fast twitch skeletal muscle. Ryanodine binding was localized to junctional cardiac SR as determined by [3H]ryanodine binding. Scatchard analysis of the binding data showed two types of binding (high affinity, Kd approximately 7.9 nM; low affinity, Kd approximately 1 microM), contrasting with skeletal junctional terminal cisternae where only one site with Kd of approximately 50 nM was observed. The ruthenium red enhancement of Ca2+ loading rate in junctional cardiac SR was blocked by pretreatment with low concentrations of ryanodine as reported for junctional terminal cisternae of skeletal muscle SR. The Ca2+ loading rate of junctional cardiac SR was enhanced by preincubation with high concentrations of ryanodine. The apparent inhibition constant (Ki approximately 7 nM) and stimulation constant (Km approximately 1.1 microM) for ryanodine on junctional SR corresponded to the Kd for high affinity binding (Kd approximately 7.9 nM) and low affinity binding (Kd approximately 1.1 microM), respectively. These results suggest that high affinity ryanodine binding locks the Ca2+ release channels in the open state and that low affinity binding closes the Ca2+ release channels of the junctional cardiac SR. The characteristics of the Ca2+ release channels of junctional cardiac SR appear to be similar to that of skeletal muscle SR, but the Ca2+ release channels of cardiac SR are more sensitive to ryanodine.     

Dihydropyridine receptor alpha subunits in normal and dysgenic muscle in vitro: expression of alpha 1 is required for proper targeting and distribution of alpha 2

We have studied the subcellular distribution of the alpha 1 and alpha 2 subunits of the skeletal muscle dihydropyridine (DHP) receptor with immunofluorescence labeling of normal and dysgenic (mdg) muscle in culture. In normal myotubes both alpha subunits were localized in clusters associated with the T-tubule membranes of longitudinally as well as transversely oriented T-tubules. The DHP receptor-rich domains may represent the sites where triad junctions with the sarcoplasmic reticulum are being formed. In cultures from dysgenic muscle the alpha 1 subunit was undetectable and the distribution patterns of the alpha 2 subunit were abnormal. The alpha subunit did not form clusters nor was it discretely localized in the T-tubule system. Instead, alpha 2 was found diffusely distributed in parts of the T-system, in structures in the perinuclear region and in the plasma membrane. These results suggest that an interaction between the two alpha subunits is required for the normal distribution of the alpha 2 subunit in the T-tubule membranes. Spontaneous fusion of normal non-muscle cells with dysgenic myotubes resulted in a regional expression of the alpha 1 polypeptide near the foreign nuclei, thus defining the nuclear domain of a T-tubule membrane protein in multi-nucleated muscle cells. Furthermore, the normal intracellular distribution of the alpha 2 polypeptide was restored in domains containing a foreign "rescue" nucleus; this supports the idea that direct interactions between the DHP receptor alpha 1 and alpha 2 subunits are involved in the organization of the junctional T-tubule membranes.     

Sequential docking, molecular differentiation, and positioning of T-Tubule/SR junctions in developing mouse skeletal muscle.

Skeletal muscle Ca(2+) release units (CRUs) are junctions of the surface membrane/T-tubule system and the sarcoplasmic reticulum (SR) that function in excitation-contraction coupling. They contain high concentrations of dihydropyridine receptors (DHPRs) in the T-tubules and of ryanodine receptors (RyR) in the SR and they are positioned at specific locations in the sarcomere. In order to characterize the sequence of developmental steps leading to the specific molecular and structural organization of CRUs, we applied a range of imaging techniques that allowed us to follow the differentiation of the membrane compartments and the expression of junctional proteins in developing mouse diaphragm muscle. We find that docking of the two membrane systems precedes the incorporation of the RyRs into the junctions, and that T-tubule/SR junctions are formed and positioned at the I-A interface at a stage when the orientation of T-tubule is predominantly longitudinal. Thus, the sequence of developmental events is first the docking of T-tubules and SR, secondly the incorporation of RyR in the junctions, thirdly the positioning of the junctions in the sarcomere, and only much later the transverse orientation of the T-tubules. These sequential stages suggests an order of inductive processes for the molecular differentiation and structural organization of the CRUs in skeletal muscle development.     

A genetic model for the study of abnormal nerve-muscle interactions at the level of excitation-contraction coupling: the mutation muscular dysgenesis

Excitation-contraction in muscle fibers are coupled through a complex mechanism involving multiproteic components located at a specialized cellular site, the triadic junction. Triads in normal muscle fiber result from the apposition of sarcoplasmic reticulum citernae and T-tubule and possess strikingly organized ultrastructural elements, bridging both types of membranes, the "junctional feet". Muscular dysgenesis in the mouse is characterized by total muscle inactivity in the developing skeletal muscles due to excitation-contraction uncoupling. Triads have been found to be disorganized with no "junctional feet" and dihydropyridine (DHP) binding sites are decreased with no slow Ca2+ currents, suggesting a basic defect in the excitation-contraction coupling machinery itself. We may hypothesize that muscular dysgenesis results in a marked defect in a functional protein involved in the morphogenesis of the triad and/or directly involved in Ca2+ release for contraction.     

Shape, size, and distribution of Ca(2+) release units and couplons in skeletal and cardiac muscles.

Excitation contraction (e-c) coupling in skeletal and cardiac muscles involves an interaction between specialized junctional domains of the sarcoplasmic reticulum (SR) and of exterior membranes (either surface membrane or transverse (T) tubules). This interaction occurs at special structures named calcium release units (CRUs). CRUs contain two proteins essential to e-c coupling: dihydropyridine receptors (DHPRs), L-type Ca(2+) channels of exterior membranes; and ryanodine receptors (RyRs), the Ca(2+) release channels of the SR. Special CRUs in cardiac muscle are constituted by SR domains bearing RyRs that are not associated with exterior membranes (the corbular and extended junctional SR or EjSR). Functional groupings of RyRs and DHPRs within calcium release units have been named couplons, and the term is also loosely applied to the EjSR of cardiac muscle. Knowledge of the structure, geometry, and disposition of couplons is essential to understand the mechanism of Ca(2+) release during muscle activation. This paper presents a compilation of quantitative data on couplons in a variety of skeletal and cardiac muscles, which is useful in modeling calcium release events, both macroscopic and microscopic ("sparks").     

Localization of the oxytocin receptor in the plasma membrane of rat myometrium.

The distribution of [3H]oxytocin binding sites among various subcellular fractions of rat myometrium paralleled the distribution of 5'-nucleotidase, a plasma membrane marker enzyme, but not of NADPH-cytochrome c reductase or succinate-cytochrome c reductase, which are endoplasmic reticulum and mitochondrial marker enzymes respectively. [3H]Oxytocin binding to the most enriched plasma membrane fraction showed the degree of selectivity with respect to hormone analogues that is expected for the oxytocin receptor. The binding of oxytocin to this fraction showed an apparent Kd of 1.98 X 10(-9) M and a capacity of 1.28 pmol mg-1. It is concluded that the oxytocin receptor is located on the plasma membrane of the smooth muscle cells of the rat uterus.     

Triadic proteins of skeletal muscle

Biochemical approaches toward understanding the mechanism of muscle excitation have in recent years been directed to identification and isolation of proteins of the triad junction. The principal protein described—the junctional foot protein (JFP)—was initially identified by morphological criteria and isolated using antibody-affinity chromatography. Subsequently this protein was described as the ryanodine receptor. It has been isolated and incorporated into lipid bilayers as a cation channel. This in its turn has directed attention toward the transverse (T)-tubular junctional constituents. Three approaches employing the JFP as a probe toward identifying these moieties on the T-tubule are described here. The binding of the JFP to the dihydropyridine receptor, which has been hypothesized to be the voltage sensor in excitation-contraction coupling, is also discussed. The detailed architecture and function of T-tubular proteins remain to be resolved.Abbreviations DHP dihydropyridine - GAPD glyceraldehyde 3-phosphate dehydrogenase - IP₃ inositol 1,4,5-trisphosphate - JFP junctional foot protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - SR sarcoplasmic reticulum - TC terminal cisterna - T-tubule transverse tubule     

The structure of Ca(2+) release units in arthropod body muscle indicates an indirect mechanism for excitation-contraction coupling

The relative disposition of ryanodine receptors (RyRs) and L-type Ca(2+) channels was examined in body muscles from three arthropods. In all muscles the disposition of ryanodine receptors in the junctional gap between apposed SR and T tubule elements is highly ordered. By contrast, the junctional membrane of the T tubule is occupied by distinctive large particles that are clustered within the small junctional domain, but show no order in their arrangement. We propose that the large particles of the junctional T tubules represent L-type Ca(2+) channels involved in excitation-contraction (e-c) coupling, based on their similarity in size and location with the L-type Ca(2+) channels or dihydropyridine receptors (DHPRs) of skeletal and cardiac muscle. The random arrangement of DHPRs in arthropod body muscles indicates that there is no close link between them and RyRs. This matches the architecture of vertebrate cardiac muscle and is in keeping with the similarity in e-c coupling mechanisms in cardiac and invertebrate striated muscles.     

COORDINATED DEVELOPMENT OF THE SARCOPLASMIC RETICULUM AND T SYSTEM DURING POSTNATAL DIFFERENTIATION OF RAT SKELETAL MUSCLE

An electron microscope study has been carried out on rat psoas muscle, during the early postnatal stages of development. Among the several subcellular components, the sarcotubular system undergoes the most striking modifications during this period. In muscle fibers of the newborn rat, junctional contacts between the T system and the SR are sparse and are, mostly, longitudinally or obliquely oriented. The T tubules do not penetrate deeply into the muscle cell, as indicated by the predominantly peripheral location of the triads and the persistence, at these stages of development, of a highly branched subsarcolemmal system of tubules. Diadic associations of junctional SR elements with the plasma membrane are also occasionally observed. The early SR elaborations incompletely delineate the myofibrils, at both the A- and I-band level. Longitudinal sections show irregularly oriented SR tubules, running continuously over successive sarcomeres. Flattened junctional cisterns filled with granular material are sparse and laterally interconnected, at circumscribed sites, with the SR tubules. Between 1 and 2 wk postpartum, transversal triadic contacts are extensively established, at the A-I band level, and the SR network differentiates into two portions in register with the A and I band, respectively. At 10–15 days after birth, the SR provides a transversely continuous double sheet around the myofibrils at the I-band level, whereas it forms a single discontinuous layer at the A-band level. The relationship that these morphological modifications of the sarcotubular system may bear to previously described biochemical and physiological changes of rat muscle fibers after birth is discussed.     

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.     

The sarcoplasmic reticulum: an organized patchwork of specialized domains

The sarcoplasmic reticulum (SR) of skeletal muscle cells is a convoluted structure composed of a variety of tubules and cisternae, which share a continuous lumen delimited by a single continuous membrane, branching to form a network that surrounds each myofibril. In this network, some specific domains basically represented by the longitudinal SR and the junctional SR can be distinguished. These domains are mainly dedicated to Ca²⁺ homeostasis in relation to regulation of muscle contraction, with the longitudinal SR representing the sites of Ca²⁺ uptake and storage and the junctional SR representing the sites of Ca²⁺ release. To perform its functions, the SR takes contact with other cellular elements, the sarcolemma, the contractile apparatus and the mitochondria, giving rise to a number of interactions, most of which are still to be defined at the molecular level. This review will describe some of the most recent advancements in understanding the organization of this complex network and its specific domains. Furthermore, we shall address initial evidence on how SR proteins are retained at distinct SR domains.     

Disposition of membranes and the entry of haemolymph-borne ferritin in flight muscle fibers of the fly Phormia regina

The disposition of the surface plasma membrane and its inwardly directed derivatives, corresponding to the T-system of other fibers, and of the corresponding sarcoplasmic reticulum (SR) elements has been examined in a dipteran asynchronous flight muscle (Photmia regina). The presence of uniaxonal neuromuscular junctions within clefts approaching the center of the fiber is described. The most conspicuous SR component is present in the dyads adjoining plasma membrane derivatives, but it is also sparsely represented elsewhere. The accessibility of the plasma membrane-limited compartments within the fiber to the ambient haemolymph, in the living insect, has been investigated by tracing the distribution of ferritin by the circulatory system. The proportion of fiber volume occupied by the T-system and SR components in asynchronous and synchronous muscle is compared and the functional implications of these proportions is discussed.     

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.