Altered Inactivation of Ca2+ Current and Ca2+ Release in Mouse Muscle Fibers Deficient in the DHP receptor γ1 subunit

Functional impacts of the skeletal muscle-specific Ca2+ channel subunit γ1 have previously been studied using coexpression with the cardiac α1C polypeptide in nonmuscle cells and primary-cultured myotubes of γ1-deficient mice. Data from single adult muscle fibers of γ−/− mice are not yet available. In the present study, we performed voltage clamp experiments on enzymatically isolated mature muscle fibers of the m. interosseus obtained from γ+/+ and γ−/− mice. We measured L-type Ca2+ inward currents and intracellular Ca2+ transients during 100-ms step depolarizations from a holding potential of −80 mV. Ratiometric Ca2+ transients were analyzed with a removal model fit approach to calculate the flux of Ca2+ from the sarcoplasmic reticulum. Ca2+ current density, Ca2+ release flux, and the voltage dependence of activation of both Ca2+ current and Ca2+ release were not significantly different. By varying the holding potential and recording Ca2+ current and Ca2+ release flux induced by 100-ms test depolarizations to +20 mV, we studied quasi-steady-state properties of slow voltage–dependent inactivation. For the Ca2+ current, these experiments showed a right-shifted voltage dependence of inactivation. Importantly, we could demonstrate that a very similar shift occurred also in the inactivation curve of Ca2+ release. Voltages of half maximal inactivation were altered by 16 (current) and 14 mV (release), respectively. Muscle fiber bundles, activated by elevated potassium concentration (120 mM), developed about threefold larger contracture force in γ−/− compared with γ+/+. This difference was independent of the presence of extracellular Ca2+ and likely results from the lower sensitivity to voltage-dependent inactivation of Ca2+ release. These results demonstrate a specific alteration of voltage-dependent inactivation of both Ca2+ entry and Ca2+ release by the γ1 subunit of the dihydropyridine receptor in mature muscle fibers of the mouse

Functional Interaction of Ca(V) Channel Isoforms with Ryanodine Receptors Studied in Dysgenic Myotubes

The L-type Ca(2+) channels Ca(V)1.1 (α(1S)) and Ca(V)1.2 (α(1C)) share properties of targeting but differ by their mode of coupling to ryanodine receptors in muscle cells. The brain isoform Ca(V)2.1 (α(1A)) lacks ryanodine receptor targeting. We studied these three isoforms in myotubes of the α(1S)-deficient skeletal muscle cell line GLT under voltage-clamp conditions and estimated the flux of Ca(2+) (Ca(2+) input flux) resulting from Ca(2+) entry and release. Surprisingly, amplitude and kinetics of the input flux were similar for α(1C) and α(1A) despite a previously reported strong difference in responsiveness to extracellular stimulation. The kinetic flux characteristics of α(1C) and α(1A) resembled those in α(1S)-expressing cells but the contribution of Ca(2+) entry was much larger. α(1C) but not α(1A)-expressing cells revealed a distinct transient flux component sensitive to sarcoplasmic reticulum depletion by 30 μM cyclopiazonic acid and 10 mM caffeine. This component likely results from synchronized Ca(2+)-induced Ca(2+) release that is absent in α(1A)-expressing myotubes. In cells expressing an α(1A)-derivative (α(1)Aas(1592-clip)) containing the putative targeting sequence of α(1S), a similar transient component was noticeable. Yet, it was considerably smaller than in α(1C), indicating that the local Ca(2+) entry produced by the chimera is less effective in triggering Ca(2+) release despite similar global Ca(2+) inward current density