title: Excitation contraction uncoupling by high intracellular [Ca2+] in frog skeletal muscle: a voltage clamp study.
abstract: Raising the intracellular [Ca2+] ([Ca2+]i) was previously found to produce uncoupling between the electrical depolarization of the transverse tubules and contraction in skinned muscle fibers. Here we study the effect of elevated [Ca2+]iin voltage clamped cut fibers of frog skeletal muscle to establish how the charge movement, a measure of the activation of the dihydropyridine receptors (DHPR)-voltage sensors, and Ca2+release, a consequence of the opening of the ryanodine receptor (RyR)-release channels, were affected. [Ca2+]iwas raised by various procedures (pharmacological release from the sarcoplasmic reticulum, application of high [Ca2+]iintracellular solution, permeabilization of the plasma membrane by a Ca2+ionophore) all of which produced impairment of excitation-contraction coupling. The charge movement was reduced from 20.2 ± 1.24 to 9.9 ± 0.94 nC/μF meanwhile the Ca2+release flux was reduced from 13.5 + 0.7 to 2.2 ± 0.3 μM/ms (n = 33). This suggests that a significant fraction of the DHPRs that remained functional, could not activate RyRs, and were therefore presumably disconnected. These results are broadly consistent with the original reports in skinned fibers. Uncoupling was prevented by the addition to the intracellular solution of the protease inhibitor leupeptin. In approximately 40 % of the uncoupled cells we observed that the [Ca2+]itransient continued to rise after the voltage clamp pulse was turned off. This loss of control by membrane voltage suggests that the uncoupled release channels might have another mechanism of activation, likely by Ca2+.
authors.affiliation: Departamento de Biofísica, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, CP11800, Montevideo, Uruguay.
authors.forename: J Fernando
authors.initials: JF
authors.lastname: Olivera
authors.affiliation: Departamento de Biofísica, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, CP11800, Montevideo, Uruguay. gpizarro@fmed.edu.uy.
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Pizarro
mesh_terms: D000818:Animals; D001001:Anura; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D056966:Excitation Contraction Coupling / Q000502:physiology*; D018482:Muscle, Skeletal / Q000378:metabolism*; D018408:Patch-Clamp Techniques / Q000379:methods*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*
chemical_list: D020746:Calcium Channels, L-Type; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium
keywords: Dihydropyridine receptor; Excitation–contraction coupling; Ryanodine receptor; Skeletal muscle