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

The role of elevated intracellular calcium concentration ([Ca2+]) in activating calcium release from the sarcoplasmic reticulum (SR) was studied in skeletal muscle fibers microinjected with strong calcium buffers. After the injection of 3.8 +/- 0.5 mM (mean +/- S.E. of mean, n = 16) BAPTA (1,2-bis[o-aminophenoxy]ethane- N,N,N',N'-tetraacetic acid) or 2.2-2.8 mM fura-2 the normal increase in [Ca2+] during a depolarizing pulse was virtually eliminated. Even though calcium was released from the SR the kinetics of this release were markedly altered: the extensive buffering selectively eliminated the early peak component of SR calcium release with no effect on the maintained steady level. Microinjections of similar volumes but with low concentrations of fura-2 had no significant effect on the release waveform. The calcium released by voltage-dependent activation during depolarization may thus be involved in activating further calcium release, that is, in a calcium-induced calcium release mechanism.

Indo-1 fluorescence signals were measured from one extremity of enzymatically isolated skeletal muscle fibers of mice. An original and simple method was developed to allow the measurements to be made under voltage-clamp control: the major part of a single fiber was embedded in silicone grease, so that only a short portion of one end of the fiber, from which the fluorescence measurements were taken, was in contact with the external solution. Membrane potential was held and varied by using a patch-clamp amplifier in whole-cell configuration with a single microelectrode, the tip of which was inserted across the silicone grease within the insulated portion of the fiber. In response to 100-ms depolarizing command pulses to voltages more positive than -40 mV (from a holding potential of -80 mV), clear changes in fluorescence were qualitatively observed to feature a time course of rise and decay expected from a change in intracellular calcium concentration ([Ca2+]i) due to voltage-dependent sarcoplasmic reticulum (SR) calcium release. Although the peak [Ca2+]i elicited by a 100-ms depolarization at 0 or +10 mV varied from fiber to fiber, it could clearly reach a value high enough to saturate Indo-1. The overall results show that this method represents an efficient way of measuring depolarization-induced [Ca2+]i changes in enzymatically dissociated skeletal muscle fibers.

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.