Excitation‐contraction coupling in skeletal muscle of a mouse lacking the dihydropyridine receptor subunit γ1

Abstract

The transverse tubules of skeletal muscle cells contain a high density of DHP binding sites (reviewed by Lamb, 1992). Two functions have been assigned to the DHPbinding proteins (DHP receptors): the activation of a Ca 2+ conductance (L-type Ca 2+ current) with exceptionally slow kinetics, and the activation of intracellular release of Ca 2+ from the sarcoplasmic reticulum (SR) (reviewed in Melzer et al. 1995). The latter process is probably the result of direct interaction between DHP receptor and Ca 2+ -release channels (ryanodine receptors) (reviewed in Melzer & Dietze, 2001) and does not depend on Ca 2+ entering the cell during the depolarization (Spiecker et al. 1979; Brum et al. 1988; Dirksen & Beam, 1999 & De Waard, 1998). a 1S and b have been shown to be essential for E-C coupling (Beam et al. 1986;Gregg et al. 1996) and their elimination is lethal. The fact that the y subunit (y1 isoform) could only be detected in skeletal muscle also suggests a role for this polypeptide that is important for muscle function (Jay et al. 1990;Powers et al. 1993;Wissenbach et al. 1998). y1 is a transmembrane protein of 32 kDa (222 amino acid residues) containing four putative membrane-spanning segments (Bosse et al. 1990; Jay et al. 1990). Functional studies of the y1 subunit have up to now been restricted to measuring transmembrane ionic currents. The effects of y1 on Ca 2+ or Ba 2+ inward currents have been investigated by coexpression of the subunit with the cardiac muscle a 1C subunit in heterologous expression systems (Singer et al. 1991;Wei et al. 1991;Lerche et al. 1996; Eberst et al. 1997;Sipos et al. 2000). An alternative approach to studying the role of subunits is the use of specific knockout systems. A recently generated y1-deficient Excitation-contraction coupling in skeletal muscle of a mouse lacking the dihydropyridine receptor subunit y1 1. In skeletal muscle, dihydropyridine (DHP) receptors control both Ca 2+ entry (L-type current) and internal Ca 2+ release in a voltage-dependent manner. Here we investigated the question of whether elimination of the skeletal muscle-specific DHP receptor subunit y1 affects excitation-contraction (E-C) coupling. We studied intracellular Ca 2+ release and force production in muscle preparations of a mouse deficient in the y1 subunit (y_/_). The rate of internal Ca2+ release at large depolarization (+20 mV) was determined in voltageclamped primary-cultured myotubes derived from satellite cells of adult mice by analysing fura-2 fluorescence signals and estimating the concentration of free and bound Ca 2+. On average, y_/_ cells showed an increase in release of about one-third of the control value and no alterations in the time course.3. Voltage of half-maximal activation (V 1/2 ) and voltage sensitivity (k) were not significantly different in y_/_ myotubes, either for internal Ca 2+ release activation or for the simultaneously measured L-type Ca 2+ conductance. The same was true for maximal Ca 2+ inward current and conductance.4. Contractions evoked by electrical stimul...

γ1 Subunit Interactions within the Skeletal Muscle L-type Voltage-gated Calcium Channels

“…␣ 2 ␦ shows a punctate or diffused pattern in mdg mice (33), whereas ␣ 1 1.1 is reduced or absent in ␤ 1 null mice (10). Recent studies have indicated that, unlike ␤ 1 (10), ␥ 1 does not have a major role in membrane trafficking of the ␣ 1 1.1 subunit as assessed by gating current measurements (21) or in EC coupling (35). Our results clearly demonstrate that the first half of the ␥ 1 subunit that allows subunit interaction also allows the restoration of L-type currents in ␥ 1 null muscle.…”

Stac3 has a direct role in skeletal muscle-type excitation–contraction coupling that is disrupted by a myopathy-causing mutation

“…Thus, KO of γ 1 (4,5) or knockdown of α 2 -δ 1 (6, 7) does not have major effects on the EC coupling and channel functions of Ca V 1.1, whereas KO of β 1a causes the loss of EC coupling (8). In part, this is because β 1a is required for efficient trafficking of Ca V 1.1 to the plasma membrane (9).…”

“…Thus, we hypothesized that RyR1 somehow relieves the inhibitory effect of another protein that is present in myotubes and absent in tsA201 cells. An obvious candidate is the remaining Ca V 1.1 auxiliary subunit, γ 1 , which we had omitted in the previous experiments because its KO was reported to have only modest effects on Ca 2+ currents in mouse skeletal muscle (4,5,17). Fig.…”

Proper Restoration of Excitation-Contraction Coupling in the Dihydropyridine Receptor β1-null Zebrafish Relaxed Is an Exclusive Function of the β1a Subunit

“…While gene knock-out of the DHPR ␥ 1 subunit (8,9) and small interfering RNA knockdown of the DHPR ␣ 2 ␦-1 subunit (10 -12) have indicated that neither subunit is essential for coupling of the DHPR with RyR1, the lack of the ␣ 1S or of the intracellular ␤ 1a subunit is incompatible with EC coupling and accordingly null model mice die perinatally due to asphyxia (13,14). ␤ subunits of voltage-gated Ca 2ϩ channels were repeatedly shown to be responsible for the facilitation of ␣ 1 membrane insertion and to be potent modulators of ␣ 1 current kinetics and voltage dependence (15,16).…”