title: Properties of calcium currents and contraction in cultured rat diaphragm muscle.
abstract: The characterization of calcium currents and contraction simultaneously measured in cultured rat diaphragm muscle cells was carried out in the present study. Whole-cell patch-clamp experiments were designed to further elucidate the mechanism of excitation-contraction (E-C) coupling in diaphragm which, though generally considered a skeletal-type muscle, has been reported to exhibit properties indicative of a cardiac-like E-C coupling mechanism. Normalized current/voltage (I/V) curves for two concentrations of external calcium (2.5 and 5 mM) were obtained from diaphragm myoballs. Both curves showed peaks corresponding to the activation of a T-type calcium current and a dihydropyridine-sensitive L-type calcium current. The normalized curve for the voltage dependence of the activation of contraction in diaphragm myoballs followed a typical Boltzmann-type relationship to the peak of contraction. Thereafter, the curve declined in a manner that was more pronounced in diaphragm compared to that measured in additional experiments using cultured rat limb muscle myoballs. This effect could be interpreted in terms of a more pronounced participation of the L-type current in E-C coupling in cultured diaphragm muscle. An increased likelihood of cultured diaphragm muscle to undergo depletion of sarcoplasmic reticular calcium stores during repetitive stimulation, or a heightened propensity for the voltage sensor for E-C coupling in diaphragm to enter the inactive state could also explain this effect. Maximal contractile activity was only slightly affected when the L-type current was blocked by externally applied cadmium (2 mM) or cobalt (3 mM), suggesting that a pronounced calcium-current-dependent component of contraction is unlikely in cultured diaphragm muscle. These results show that T- and L-type calcium channels are expressed in cultured rat diaphragm muscle cells and that, in contrast to cardiac muscle, the entry of calcium ions via L-type voltage-dependent calcium channels is not a prerequisite for contraction. Differences in the voltage sensitivity of contraction, observed at depolarized membrane potentials in cultured rat diaphragm and limb muscle cells, suggest that the voltage sensor for E-C coupling in diaphragm might more readily enter an inactivated configuration - possibly by a mechanism which is dependent on the concentration of external calcium.
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000378:metabolism* / Q000502:physiology; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003964:Diaphragm / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D004594:Electrophysiology; D006614:Hindlimb / Q000187:drug effects / Q000502:physiology; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D018482:Muscle, Skeletal / Q000187:drug effects / Q000502:physiology; D009132:Muscles / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar
chemical_list: D015220:Calcium Channels; D002413:Cations, Divalent