Skeletal muscle contraction is triggered by the excitation-contraction (E-C) coupling machinery residing at the triad, a membrane structure formed by the juxtaposition of T-tubules and sarcoplasmic reticulum (SR) cisternae. The formation and maintenance of this structure is key for muscle function but is not well characterized. We have investigated the mechanisms leading to X-linked myotubular myopathy (XLMTM), a severe congenital disorder due to loss of function mutations in the MTM1 gene, encoding myotubularin, a phosphoinositide phosphatase thought to have a role in plasma membrane homeostasis and endocytosis. Using a mouse model of the disease, we report that Mtm1-deficient muscle fibers have a decreased number of triads and abnormal longitudinally oriented T-tubules. In addition, SR Ca 2؉ release elicited by voltageclamp depolarizations is strongly depressed in myotubularin-deficient muscle fibers, with myoplasmic Ca 2؉ removal and SR Ca 2؉ content essentially unaffected. At the molecular level, Mtm1-deficient myofibers exhibit a 3-fold reduction in type 1 ryanodine receptor (RyR1) protein level. These data reveal a critical role of myotubularin in the proper organization and function of the E-C coupling machinery and strongly suggest that defective RyR1-mediated SR Ca 2؉ release is responsible for the failure of muscle function in myotubular myopathy. myotubular myopathy ͉ triad

Caenorhabditis elegans is a powerful model system widely used to investigate the relationships between genes and complex behaviors like locomotion. However, physiological studies at the cellular level have been restricted by the difficulty to dissect this microscopic animal. Thus, little is known about the properties of body wall muscle cells used for locomotion. Using in situ patch clamp technique, we show that body wall muscle cells generate spontaneous spike potentials and develop graded action potentials in response to injection of positive current of increasing amplitude. In the presence of K+ channel blockers, membrane depolarization elicited Ca2+ currents inhibited by nifedipine and exhibiting Ca2+-dependent inactivation. Our results give evidence that the Ca2+ channel involved belongs to the L-type class and corresponds to EGL-19, a putative Ca2+ channel originally thought to be a member of this class on the basis of genomic data. Using Ca2+ fluorescence imaging on patch-clamped muscle cells, we demonstrate that the Ca2+ transients elicited by membrane depolarization are under the control of Ca2+ entry through L-type Ca2+ channels. In reduction of function egl-19 mutant muscle cells, Ca2+ currents displayed slower activation kinetics and provided a significantly smaller Ca2+ entry, whereas the threshold for Ca2+ transients was shifted toward positive membrane potentials.

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The cellular mechanisms responsible for the progressive skeletal muscle degeneration that characterizes the disease are still debated. One hypothesis suggests that the resting sarcolemmal permeability for Ca 2؉ is increased in dystrophic muscle, leading to Ca 2؉ accumulation in the cytosol and eventually to protein degradation. However, more recently, this hypothesis was challenged seriously by several groups that did not find any significant increase in the global intracellular Ca 2؉ in muscle from mdx mice, an animal model of the human disease. In the present study, using plasma membrane Ca 2؉ -activated K ؉ channels as subsarcolemmal Ca 2؉ probe, we tested the possibility of a Ca 2؉ accumulation at the restricted subsarcolemmal level in mdx skeletal muscle fibers. Using the cell-attached configuration of the patch-clamp technique, we demonstrated that the voltage threshold for activation of high conductance Ca 2؉ -activated K ؉ channels is significantly lower in mdx than in control muscle, suggesting a higher subsarcolemmal [Ca 2؉ ]. In inside-out patches, we showed that this shift in the voltage threshold for high conductance Ca 2؉ -activated K ؉ channel activation could correspond to a Ϸ3-fold increase in the subsarcolemmal Ca 2؉ concentration in mdx muscle. These data favor the hypothesis according to which an increased calcium entry is associated with the absence of dystrophin in mdx skeletal muscle, leading to Ca 2؉ overload at the subsarcolemmal level.

The myotendinous junction (MTJ) is the major site of force transfer in skeletal muscle, and defects in its structure correlate with a subset of muscular dystrophies. Col22a1 encodes the MTJ component collagen XXII, the function of which remains unknown. Here, we have cloned and characterized the zebrafish col22a1 gene and conducted morpholino-based loss-of-function studies in developing embryos. We showed that col22a1 transcripts localize at muscle ends when the MTJ forms and that COLXXII protein integrates the junctional extracellular matrix. Knockdown of COLXXII expression resulted in muscular dystrophy-like phenotype, including swimming impairment, curvature of embryo trunk/tail, strong reduction of twitch-contraction amplitude and contraction-induced muscle fiber detachment, and provoked significant activation of the survival factor Akt. Electron microscopy and immunofluorescence studies revealed that absence of COLXXII caused a strong reduction of MTJ folds and defects in myoseptal structure. These defects resulted in reduced contractile force and susceptibility of junctional extracellular matrix to rupture when subjected to repeated mechanical stress. Co-injection of subphenotypic doses of morpholinos against col22a1 and genes of the major muscle linkage systems showed a synergistic gene interaction between col22a1 and itga7 (α7β1 integrin) that was not observed with dag1 (dystroglycan). Finally, pertinent to a conserved role in humans, the dystrophic phenotype was rescued by microinjection of recombinant human COLXXII. Our findings indicate that COLXXII contributes to the stabilization of myotendinous junctions and strengthens skeletal muscle attachments during contractile activity.

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Odontoblasts are responsible for the dentin formation. They are suspected to play a role in tooth pain transmission as sensor cells because of their close relationship with nerve, but this role has never been evidenced. We demonstrate here that human odontoblasts in vitro produce voltage-gated tetrodotoxin-sensitive Na ؉ currents in response to depolarization under voltage clamp conditions and are able to generate action potentials. Odontoblasts express neuronal isoforms of ␣2 and ␤2 subunits of sodium channels. Co-cultures of odontoblasts with trigeminal neurons indicate a clustering of ␣2 and ␤2 sodium channel subunits and, at the sites of cell-cell contact, a co-localization of odontoblasts ␤2 subunits with peripherin. In vivo, sodium channels are expressed in odontoblasts. Ankyrin G and ␤2 co-localize, suggesting a link for signal transduction between axons and odontoblasts. Evidence for excitable properties of odontoblasts and clustering of key molecules at the site of odontoblast-nerve contact strongly suggest that odontoblasts may operate as sensor cells that initiate tooth pain transmission.

Odontoblasts form a layer of cells responsible for the dentin formation and possibly mediate early stages of sensory processing in teeth. Several classes of ion channels have previously been identified in the odontoblast or pulp cell membrane, and it is suspected that these channels assist in these events. This study was carried out to characterize the K Ca channels on odontoblasts fully differentiated in vitro using the patch clamp technique and to investigate the HSLO gene expression encoding the ␣-subunit of these channels on odontoblasts in vivo. In inside-out patches, K Ca channels were identified on the basis of their K ؉ selectivity, conductance, voltage, and Ca 2؉ dependence. In cell-attached patches, these channels were found to be activated by application of a negative pressure as well as an osmotic shock. By reverse transcription-polymerase chain reaction, a probe complementary to K Ca ␣-subunit mRNA was constructed and used for in situ hybridization on human dental pulp samples. Transcripts were expressed in the odontoblast layer. The use of antibodies showed that the K Ca channels were preferentially detected at the apical pole of the odontoblasts. These channels could be involved in mineralization processes. Their mechanosensitivity suggests that the fluid displacement within dentinal tubules could be transduced into electrical cell signals.

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