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

Extensive studies performed in nonexcitable cells and expression systems have shown that type 1 transient receptor potential canonical (TRPC1) channels operate mainly in plasma membranes and open through phospholipase C-dependent processes, membrane stretch, or depletion of Ca 2؉ stores. In skeletal muscle, it is proposed that TRPC1 channels are involved in plasmalemmal Ca 2؉ influx and stimulated by store depletion or membrane stretch, but direct evidence for TRPC1 sarcolemmal channel activity is not available. We investigated here the functional role of TRPC1 using an overexpressing strategy in adult mouse muscle fibers. Immunostaining for endogenous TRPC1 revealed a striated expression pattern that matched sarcoplasmic reticulum ( Transient receptor potential canonical 1 (TRPC1) 2 proteins consist of nonselective cation channels expressed in a great variety of multicellular organisms (1, 2). Extensive studies performed in nonexcitable cells or involving heterologously expressed channels have shown that TRPC1 channels operate mainly in homo-or heteromeric association with other TRPC isoforms as Ca 2ϩ -permeable channels in the plasma membrane. Parameters that control TRPC1 channel opening remain controversial. TRPC1 channels could be activated by direct interaction with endoplasmic reticulum inositol trisphosphate receptors or at least through phospholipase C-dependent processes, membrane stretch, or depletion of intracellular Ca 2ϩ stores (3). Much less data are available concerning endogenous TRPC1 channels in excitable cells. In skeletal muscle, TRPC1 proteins were first shown to be expressed in the sarcolemma of muscles from dystrophin-deficient mouse (mdx), a murine model of Duchenne muscular dystrophy, and were proposed to mediate a sarcolemmal store-operated Ca 2ϩ influx in adult fibers (4) and contribute to cell migration and fusion in cultured myoblasts as store-operated or stretch-activated channels (5, 6). Gervásio et al. (7) confirmed the sarcolemmal localization of TRPC1 in mouse muscle and indicated that the sarcolemmal level of TRPC1 was slightly increased in mdx fibers. Stiber et al. (8) also described a sarcolemmal pattern of expression associated with a striated transversal pattern assumed to correspond to costamers at the level of Z discs. TRPC1 was also found to associate with skeletal muscle scaffolding proteins, including caveolin-3 and Homer1 in adult muscle fibers and dystrophin and ␣1-syntrophin in cultured myotubes (7-9).Taken together, these data suggest that skeletal muscle TRPC1 channels are involved in sarcolemmal Ca 2ϩ influx possibly stimulated by store depletion or membrane stretch, but direct evidence for the existence of a measurable Ca 2ϩ conductance supported by TRPC1 channels at resting potentials is not available. We previously showed in adult mouse muscle fibers that sarcoplasmic reticulum (SR) Ca 2ϩ depletion failed to induce any increase in the resting whole-cell conductance and inward single channel activity (10). We also demonstrated that the resting and store-operat...

Ca2+ is known to enter skeletal muscle at rest and during activity. Except for the well-characterized Ca2+ entry through L-type channels, pathways involved in these Ca2+ entries remain elusive in adult muscle. This study investigates Ca2+ influx at rest and during activity using the method of Mn2+ quenching of fura-2 fluorescence on voltage-controlled adult skeletal muscle cells. Resting rate of Mn2+ influx depended on external [Mn2+] and membrane potential. At -80 mV, replacement of Mg2+ by Mn2+ gave rise to an outward current associated with an increase in cell input resistance. Calibration of fura-2 response indicated that Mn2+ influx was too small to be resolved as a macroscopic current. Partial depletion of the sarcoplasmic reticulum induced by a train of action potentials in the presence of cyclopiazonic acid led to a slight increase in resting Mn2+ influx but no change in cell input resistance and membrane potential. Trains of action potentials considerably increased Mn2+ entry through an electrically silent pathway independent of L-type channels, which provided 24% of the global Mn2+ influx at +30 mV under voltage-clamp conditions. Within this context, the nature and the physiological role of the Ca2+ pathways involved during muscle excitation still remain open questions.