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

Abstract

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.

A retrograde signal from RyR1 alters DHP receptor inactivation and limits window Ca 2+ release in muscle fibers of Y522S RyR1 knock-in mice

“…Voltage Clamping and Data Analysis. The experimental design was as described (11,27). Briefly, isolated fibers were voltage-clamped with a two-electrode system (Axoclamp 2B, Axon Instruments) and imaged using a fluorescence microscope (Axiovert 135 TV, 40x/0.75W objective, Zeiss).…”

The auxiliary subunit γ 1 of the skeletal muscle L-type Ca 2+ channel is an endogenous Ca 2+ antagonist

“…In the skeletal muscle L-type channel, the ␥-subunit (␥ 1 ), shows the highest tissue specificity next to the ␣ 1 -polypeptide. Interestingly, ␥ 1 deficiency in voltage-clamped adult muscle fibers of gene-targeted knockout mice affected both Ca 2ϩ current and Ca 2ϩ release by shifting the steady-state availability curves to more positive potentials (12). Thus, the ␥ 1 -polypeptide exhibits a physiological effect similar to the one reported for PAA Ca 2ϩ channel antagonists.…”

“…The estimates of Ca 2ϩ release flux and of the SRluminal Ca 2ϩ concentrations are proportional to the assumed intracellular EGTA concentration (39). For the removal analysis we used the pipette concentrations of EGTA and fura-2 and subsequently scaled down the resulting flux amplitudes and SR-luminal Ca 2ϩ concentrations by a factor of 0.4 to account for mean fractional loading of the cell at the time of recording (12). The voltage-activated Ca 2ϩ permeability of the SR (in % ms Ϫ1 ) was calculated according to Gonzalez and Ríos (17) under the assumption that the slow decline in the plateau phase of the Ca 2ϩ release flux results exclusively from SR depletion.…”

“…When strong enough, steady depolarization would cause maintained Ca 2ϩ release and consequently involuntary muscle contractures. The slow inactivation process of the DHPR can be envisaged as a mechanism to counteract such effects, but it leaves a window of voltages in which steady activation is possible (12,37). Agents with the functional characteristics of Ca 2ϩ antagonists will narrow this window.…”

Exploring the function and pharmacotherapeutic potential ­of voltage-gated Ca2+channels with gene-knockout models

“…238 The shift of voltagedependent inactivation to more positive voltages was also confirmed in isolated mature muscle fibres. 239 Similar to current, γ1-deficiency also shifted the voltage-dependence of the inactivation of SR Ca 2+ release fluxes to more positive voltages. As a consequence, more channel activity should be available for triggering SR Ca 2+ release flux during prolonged depolarization but not during short twitches.…”