Sustained Release of Calcium Elicited by Membrane Depolarization in Ryanodine-Injected Mouse Skeletal Muscle Fibers

Abstract

The effect of micromolar intracellular levels of ryanodine was tested on the myoplasmic free calcium concentration ([Ca(2+)](i)) measured from a portion of isolated mouse skeletal muscle fibers voltage-clamped at -80 mV. When ryanodine-injected fibers were transiently depolarized to 0 mV, the early decay phase of [Ca(2+)](i) upon membrane repolarization was followed by a steady elevated [Ca(2+)](i) level. This effect could be qualitatively well simulated, assuming that ryanodine binds to release channels that open during depolarization and that ryanodine-bound channels do not close upon repolarization. The amplitude of the postpulse [Ca(2+)](i) elevation depended on the duration of the depolarization, being hardly detectable for pulses shorter than 100 ms, and very prominent for duration pulses of seconds. Within a series of consecutive pulses of the same duration, the effect of ryanodine produced a staircase increase in resting [Ca(2+)](i), the slope of which was approximately twice larger for depolarizations to 0 or +10 mV than to -30 or -20 mV. Overall results are consistent with the "open-locked" state because of ryanodine binding to calcium release channels that open during depolarization. Within the voltage-sensitive range of calcium release, increasing either the amplitude or the duration of the depolarization seems to enhance the fraction of release channels accessible to ryanodine.

Excitation-contraction coupling in skeletal muscle fibers from adult domestic honeybee

“…This Ca 2+ transient depression was accompanied by a progressive increase in resting Ca 2+ , due to a steady elevated level following the Ca 2+ transient decay phase following repolarization. These results were indicative of an open-locked state for the RyR in situ, a feature consistent with a usedependence effect [17]. Conversely, a higher ryanodine concentration (100 µM) than the one used in the present study was found to induce a "modest increase" of the voltage-activated Ca 2+ transients in crayfish muscle fibers, which reflects the complexity of ryanodine actions at low and high concentration [28].…”

Sarcoplasmic reticulum Ca2+ release and depletion fail to affect sarcolemmal ion channel activity in mouse skeletal muscle

“…Mice were killed by cervical dislocation before removal of the muscles. Procedures for enzymatic isolation of single muscle fibres, partial insulation of the fibres with silicone grease and intracellular microinjection were as previously described (Jacquemond, 1997; Collet et al 1999, 2004; Collet & Jacquemond, 2002). In brief, the major part of a single fibre was electrically insulated with silicone grease so that whole‐cell voltage‐clamp could be achieved on a short portion of the fibre extremity.…”

“…For these, the solution that was microinjected in the fibres also contained 1 m m of either indo‐1 or fluo‐3. The optical set‐up and the procedures used for the indo‐1 fluorescence measurements were as previously described (Jacquemond, 1997; Collet et al 1999; Collet & Jacquemond, 2002; Pouvreau et al 2004). In brief, a Nikon Diaphot epifluorescence microscope was used in diafluorescence mode.…”

Expression of the muscular dystrophy-associated caveolin-3P104L mutant in adult mouse skeletal muscle specifically alters the Ca2+ channel function of the dihydropyridine receptor

“…Procedures for enzymatic isolation of single fibres, partial insulation of the fibres with silicone grease and intracellular dye loading were as previously described [5,7,8,19]. In brief, mice were killed by cervical dislocation after halothane (Sigma Aldrich, Saint Quentin Fallavier, France) inhalation, before removal of the muscles.…”

“…Results were either expressed in terms of indo-1% saturation or in actual free Ca 2+ concentration (for details of calculation, see [10,19]). In vivo values for R min , R max and β were measured using procedures previously described [5,7]. Corresponding mean values measured in control and Cav-3 P104L -expressing fibres were 0.33±0.09, 1.29±0.15, 2.79±0.11 (n=8) and 0.31±0.12, 1.31±0.14, 2.84±0.10 (n=7), respectively.…”