Skeletal muscle L-type Ca(2+) current modulation in gamma1-deficient and wildtype murine myotubes by the gamma1 subunit and cAMP

Abstract

Modulation of the steady-state inactivation and current amplitude by the gamma1 subunit of the murine skeletal muscle L-type Ca(2+) channel were investigated using the whole-cell patch-clamp technique. Transient expression of the gamma1 subunit, but not of the gamma2 (stargazin) protein, in primary cultured myotubes from gamma1-deficient mice shifted the steady-state inactivation approximately -15 mV, thereby restoring wildtype (WT) steady-state inactivation and current amplitude. The increased Ca(2+) current amplitude in gamma1-deficient cells was abolished in myotubes from animals of 4 weeks and older whereas the positive shift in steady-state inactivation was independent of mouse age. Raising intracellular cAMP levels using the membrane-permeant analogue 8-Br-cAMP led to an increase in Ca(2+) current amplitude in WT cells to the level in gamma1-deficient myotubes. There was no effect on the current amplitude in gamma1-deficient cells or on the steady-state inactivation in either genotype. Rp-cAMPS, a competitive inhibitor of cAMP-dependent protein kinase, had no effect on the WT Ca(2+) current amplitude and steady-state inactivation, but diminished the current amplitude in gamma1-deficient myotubes without affecting the steady-state inactivation in these cells. These data show that the increased Ca(2+) influx in myotubes lacking the gamma1 subunit, due to right-shifted steady-state inactivation and increased L-type Ca(2+) current amplitude, is determined by the gamma1 subunit. The effect on current amplitude depends on the age of the mice and its cAMP-dependent modulation appears to be controlled by the gamma1 subunit.

Figure 1. Expression of the γ1 subunit in γ1-deficient myotubes restores WT L-type Ca²⁺ current amplitude and steady-state inactivation

A, representative current traces during a 400 ms depolarisation at −40 mV and at +20 mV from a WT and a γ1-deficient myotube, and from a γ1-deficient cell transfected with the γ1 subunit. The dashed line indicates zero. At −40 mV, a T-type Ca²⁺ channel is activated in the γ1-deficient cell. B, average steady-state inactivation (normalised to −100 mV prepulse potential) from WT (filled squares, n = 27), γ1-deficient cells (filled circles, n = 36) and from γ1-transfected γ1-deficient cells (open circles, n = 8). Data were fitted with a Boltzman equation. C, average I-V relationships of WT (filled squares, n = 30), γ1−/− (filled circles, n = 29) and γ1-transfected γ1-deficient (open circles, n = 9) myotubes. D, bar graphs showing the current density at +20 mV (top) and the steady-state inactivation after a prepulse of −20 mV (bottom) for WT and γ1-deficient myotubes, γ1-deficient myotubes expressing the γ1or the γ2 protein. Asterisks indicate statistical significance (P < 0.05), values in parentheses indicate number of cells.

Figure 2. Age dependence of L-type Ca²⁺ current amplitude and steady-state inactivation

A, current densities at +20 mV are plotted for WT and γ1-deficient myotubes from neonatal, 2-week-old, 4-week-old and 4-month-old mice. B, normalised steady-state inactivation after a 5 s prepulse to −20 mV for WT and γ1-deficient myotubes from neonatal, 2-week-old, 4-week-old and 4-month-old mice. Asterisks indicate statistical significance (P < 0.05), values in parentheses indicate number of cells.

Figure 3. Effects of 8-Br-cAMP on L-type Ca²⁺ current amplitude and steady-state inactivation

A, averaged I-V relationships in the presence and absence of 100 μm 8-Br-cAMP from WT (filled squares, without 8-Br-cAMP, n = 30; open squares, with 8-Br-cAMP, n = 16) and γ1-deficient (filled circles, without 8-Br-cAMP, n = 29; open circles, with 8-Br-cAMP, n = 18) myotubes. B, representative current traces at +20 mV in the absence (control) or presence of 100 μm 8-Br-cAMP from a WT and a γ1-deficient myotube. Traces of WT and γ1-deficient cells in the absence of 8-Br-cAMP same as in Fig. 1A. C, normalised steady-state inactivation for WT (filled squares, n = 27) and γ1-deficient cells (filled circles, n = 36) under control conditions and in the presence of 8-Br-cAMP (100 μm) (WT, open squares, n = 16; γ1−/−, open circles, n = 18). D, bar graph summarising the effect of 100 μm 8-Br-cAMP on the current density at +20 mV (top) and on the steady-state inactivation after a prepulse of −20 mV (bottom) for WT and γ1-deficient myotubes. Asterisks indicate statistical significance (P < 0.05), values in parentheses indicate number of cells.

Figure 4. Effects of Rp-cAMPS on L-type Ca²⁺ current amplitude and steady-state inactivation

A, averaged I-V relationships in the presence and absence of 100 μm Rp-cAMPS from WT (filled squares, without Rp-cAMPS, n = 30; open squares, with Rp-cAMPS, n = 17) and γ1-deficient (filled circles, without Rp-cAMPS, n = 29; open circles, with Rp-cAMPS, n = 20) myotubes. B, representative current traces at +20 mV in the absence (control) or presence of 100 μm Rp-cAMPS from a WT and a γ1-deficient myotube. Traces of WT and γ1-deficient cells in the absence of Rp-cAMPS same as in Fig. 1A,C, normalised steady-state inactivation for WT (filled squares, n = 27) and γ1-deficient cells (filled circles, n = 36) under control conditions and in the presence of Rp-cAMPS (100 μm) (WT, open squares, n = 18; γ1−/−, open circle, n = 18). D, bar graph summarising the effect of 100 μm Rp-cAMPS on the current density at +20 mV (top) and on the steady-state inactivation after a prepulse of −20 mV (bottom) for WT and γ1-deficient myotubes. Asterisks indicate statistical significance (P < 0.05), values in parentheses indicate number of cells.