Short-term regulation of excitation-contraction coupling by the beta1a subunit in adult mouse skeletal muscle

Abstract

The beta1a subunit of the skeletal muscle voltage-gated Ca2+ channel plays a fundamental role in the targeting of the channel to the tubular system as well as in channel function. To determine whether this cytosolic auxiliary subunit is also a regulatory protein of Ca2+ release from the sarcoplasmic reticulum in vivo, we pressure-injected the beta1a subunit into intact adult mouse muscle fibers and recorded, with Fluo-3 AM, the intracellular Ca2+ signal induced by the action potential. We found that the beta1a subunit significantly increased, within minutes, the amplitude of Ca2+ release without major changes in its time course. beta1a subunits with the carboxy-terminus region deleted did not show an effect on Ca2+ release. The possibility that potentiation of Ca2+ release is due to a direct interaction between the beta1a subunit and the ryanodine receptor was ruled out by bilayer experiments of RyR1 single-channel currents and also by Ca2+ flux experiments. Our data suggest that the beta1a subunit is capable of regulating E-C coupling in the short term and that the integrity of the carboxy-terminus region is essential for its modulatory effect.

FIGURE 1

The action of the β_1a subunit upon Ca signals. (A) The records show Fluo-3 Ca²⁺ signals associated with action potentials from a muscle fiber that was pressure injected with a control solution. (B) Ca²⁺ signals from a separate fiber to which the β_1a subunit was pressure injected. The numbers below each trace indicate time in minutes computed since microinjection was done.

FIGURE 2

The time course of action of the β_1a subunit. Circles represent mean values (± SE) of peak Ca²⁺ signals as a function of time. Values were normalized for every experiment as: Peak j / Peak a, where j is every individual peak Ca²⁺ signal value and a is the average peak value of signals recorded before pressure injection; (○) represents values of normalized records obtained from pressure-injected fibers with the control solution; (•) represents values from pressure-injected fibers with the β_1a subunit. The arrow indicates time of microinjection. Unless otherwise indicated by the number in parentheses, each symbol represents the mean value of at least five to seven separate experiments. The smooth curve is the graph of Eq. 2 at x = 125 μm and D = 6.2 × 10⁻⁸ cm² s⁻¹.

FIGURE 3

The action of the β_1a subunit on charge movement. (A) (○) Represents mean values (± SE) of charge movement as a function of membrane potential from control experiments (n = 16). The smooth curve is the best fit of Eq. 3 to the data points with Q_max = 31.3 nC/μF, V = −36.6 mV, and k = 15.0 mV. (B) (•) Represents mean values (± SE) of charge movement as a function of membrane potential in the presence of the β_1a subunit (n = 16). E_h = −100 mV. The smooth curve is the Boltzmann fit with Q_max = 30.2 nC/μF, V = −36.0 mV, and k = 13.9 mV. The insets in panels A and B show records of nonlinear currents from representative experiments. The amplitude and timescale in panel A also applies to panel B.

FIGURE 4

The action of the β_1a subunit on L-type currents. (A) The records show nonlinear currents from a control experiment at the potentials indicated with the numbers (in mV) between traces. (B) Records of membrane currents at the same potentials from a separate experiment performed in the presence of the β_1a subunit. Currents were normalized per unit capacitance. (C) The current-voltage relationship of Ca²⁺ currents. Symbols represent mean values (± SE) of peak Ca²⁺ currents from control experiments (○) (n = 21) and from experiments performed in the presence of the β_1a subunit (•) (n = 16). E_h = −80 mV. The smooth curve is the best fit of Eq. 4 to the data points with G_max = 122.5 μS/μF, V = 3.4 mV, k = 4.2 mV, and V_rev = 70.2 mV (○) and G_max = 163.6 μS/μF, V = 7.0 mV, k = 3.1 mV, and V_rev = 81.7 mV (•). (D) The voltage dependence of the normalized conductance. Mean current density values from panel C were divided by G_max × (V_m − V_rev). Smooth curves are best fits of a Boltzmann function with V = 3.4 mV and k = 4.1 mV (○) and V = 7.0 mV and k = 3.0 mV (•).

FIGURE 5

The involvement of the carboxy-terminus region of the β_1a subunit on Ca²⁺ signal potentiation. (A) The records show Fluo-3 Ca²⁺ signals associated with action potentials from a muscle fiber that was pressure injected with the full-length β_1a subunit purified from bacteria. (B) Ca²⁺ signals from a separate fiber to which a truncated form of the β_1a subunit, lacking 40 amino acids in the carboxy-terminus region, was pressure injected. The numbers below each trace indicate time in minutes computed since microinjection was carried out. The dotted lines indicate resting fluorescence and peak ΔF/F before pressure injection.

FIGURE 6

The action of the β_1a subunit on Ca²⁺ leak from SR microsomes and on single RyR1 channel function. (A) The Ca²⁺ leak rate of Ca²⁺ loaded skeletal muscle SR microsomes (50 μg) was recorded after SERCA pump blockade by 25 μM cyclopiazonic acid (CPZ). Leak was measured in different experimental conditions (□, 2.5 mM caffeine; ⋄, 5 μM ruthenium red; •, β_1a subunit; ○, truncated β_1a subunit). (B) Single RyR1 channel activity in planar lipid bilayers was monitored in the absence (control) and presence of the β_1a subunit. The charge carrier was Ca²⁺ and holding potential was 0 mV. The solution in the cytosolic chamber had 2 μM free Ca²⁺, 5.5 mM total Mg²⁺, and 7.0 mM total ATP.