Convergent regulation of skeletal muscle Ca2+ channels by dystrophin, the actin cytoskeleton, and cAMP-dependent protein kinase

Abstract

The skeletal muscle L-type Ca2+ channel (Ca(V)1.1), which is responsible for initiating muscle contraction, is regulated by phosphorylation by cAMP-dependent protein kinase (PKA) in a voltage-dependent manner that requires direct physical association between the channel and the kinase mediated through A-kinase anchoring proteins (AKAPs). The role of the actin cytoskeleton in channel regulation was investigated in skeletal myocytes cultured from wild-type mice, mdx mice that lack the cytoskeletal linkage protein dystrophin, and a skeletal muscle cell line, 129 CB3. Voltage dependence of channel activation was shifted positively, and potentiation was greatly diminished in mdx myocytes and in 129 CB3 cells treated with the microfilament stabilizer phalloidin. Voltage-dependent potentiation by strong depolarizing prepulses was reduced in mdx myocytes but could be restored by positively shifting the stimulus potentials to compensate for the positive shift in the voltage dependence of gating. Inclusion of PKA in the pipette caused a negative shift in the voltage dependence of activation and restored voltage-dependent potentiation in mdx myocytes. These results show that skeletal muscle Ca2+ channel activity and voltage-dependent potentiation are controlled by PKA and microfilaments in a convergent manner. Regulation of Ca2+ channel activity by hormones and neurotransmitters that use the PKA signal transduction pathway may interact in a critical way with the cytoskeleton and may be impaired by deletion of dystrophin, contributing to abnormal regulation of intracellular calcium concentrations in dystrophic muscle.

Fig. 1.

Voltage dependence of Ca²⁺ channel activation in mdx skeletal muscle. (A) Currents recorded from a control mouse myotube in response to 500-ms-long pulses to the indicated potentials. (B) Current traces from an mdx myotube. (C) Mean current–voltage relationships from control (•) and mdx (▪) mouse myotubes. The mean peak Ba²⁺ currents for control and mdx myocytes were not significantly different in this sample of cells (954 pA for control and 737 pA for mdx), minimizing possible contributions of series resistance to the voltage shifts measured.

Fig. 2.

Reduced voltage-dependent potentiation in mdx skeletal muscle. Ca²⁺ channel current was recorded in the whole-cell patch clamp mode from control and mdx mouse skeletal muscle myocytes. (A) Examples of Ca²⁺ channel potentiation after a conditioning prepulse in control and mdx muscle. From a holding potential of –80 mV, cells were depolarized to –20 mV for 300 ms in test pulse 1 and returned to the holding potential. A prepulse to +80 mV for 200 ms was applied, the cells were briefly repolarized to –60 mV, and Ba²⁺ currents were recorded during a second identical test pulse. Currents in test pulse 1 were normalized to the same amplitudes (control, •; mdx, ▴). To allow direct comparison of potentiation after the prepulse the currents in test pulse 2 were normalized by the same factor (control, ○; mdx, ▵). The rapidly activated and inactivated current in the “Before prepulse” trace is T-type current, which was observed in a subset of the cells. Data are representative of 10 and 12 experiments, respectively. (B) Ca²⁺ channel current–voltage relations measured before (control, •; mdx, ▴) and after (control, ○; mdx, ▵) conditioning prepulses are shown to compare channel potentiation at the indicated test potentials. Data are mean ± SEM (n = 9 for each).

Fig. 3.

Recovery of voltage-dependent potentiation in mdx myocytes by compensation for the positive shift of the voltage dependence of activation. (A) Ca²⁺ channel potentiation in mdx myocytes measured with the same protocol as in Fig. 2 but with all potentials shifted by the indicated amounts. (B) Mean potentiation (± SEM, n = 5) in mdx muscle vs. shift in membrane potential is shown.

Fig. 4.

Restoration of voltage-dependent potentiation in mdx skeletal muscle by PKA. (A) Ca²⁺ channel current–voltage relations measured before (▪) and after (□) conditioning prepulses are shown for mdx muscle to which additional catalytic subunit of PKA (2 μM) was applied through the patch pipette. (B) Mean potentiation (± SEM, n = 5–12) measured at –20 mV for control muscle, mdx muscle, and mdx muscle with 2 μM added PKA are shown. (C) Voltage dependence of Ca²⁺ channel activation is shown for control muscle (•, n = 14), mdx muscle (▾, n = 12), and mdx muscle with 2 μM added PKA (▪, n = 6).

Fig. 5.

Effect of PKA activity on voltage dependence of Ca²⁺ channel activation. (A) Mean conductance–voltage relationships in control mouse 129 CB3 myotubes (○), myotubes dialyzed intracellularly with PKA (▴), and myocytes dialyzed intracellularly with the PKA inhibitor peptide, PKI (▪). (B) Mean potentiation of Ca²⁺ channels in 129 CB₃ cells (± SEM, n = 20, 9) measured by using the voltage-clamp protocol of Fig. 2.

Fig. 6.

Recovery of potentiation in the presence of PKI by compensation for the shift in the voltage dependence of activation. (A) Examples of Ca²⁺ channel potentiation measured in PKI-treated muscle cells with increasing shift in membrane potential. Ca²⁺ channel potentiation was first measured by using the voltage-clamp protocol of Fig. 2, then all membrane potentials were shifted as indicated on the abscissa, and potentiation was measured again. (B) Mean potentiation (± SEM) vs. shift in membrane potential in the potentiation protocol (n = 13) is shown.

Fig. 7.

Effect of actin filament stabilization and destabilization. (A) Destabilization of actin filaments with cytochalasin D. Shown are mean conductance–voltage relationships as in Fig. 5 for control myocytes (○), myocytes treated with 20 μM cytochalasin D (▪), and myocytes treated with cytochalasin D and intracellularly dialyzed with PKA during recording (▴). (B) Stabilization of actin filaments with phalloidin. Shown are mean conductance–voltage relationships in control myocytes (○), myocytes treated with phalloidin (▪), and myocytes treated with phalloidin plus intracellular PKA (▴).