Role of Calcium Permeation in Dihydropyridine Receptor Function : Insights into Channel Gating and Excitation–Contraction Coupling

The skeletal and cardiac muscle dihydropyridine receptors (DHPRs) differ with respect to their rates of channel activation and in the means by which they control Ca²⁺ release from the sarcoplasmic reticulum (Adams, B.A., and K.G. Beam. 1990. FASEB J. 4:2809–2816). We have examined the functional properties of skeletal (SkEIIIK) and cardiac (CEIIIK) DHPRs in which a highly conserved glutamate residue in the pore region of repeat III was mutated to a positively charged lysine residue. Using expression in dysgenic myotubes, we have characterized macroscopic ionic currents, intramembrane gating currents, and intracellular Ca²⁺ transients attributable to these two mutant DHPRs. CEIIIK supported very small inward Ca²⁺ currents at a few potentials (from −20 to +20 mV) and large outward cesium currents at potentials greater than +20 mV. SkEIIIK failed to support inward Ca²⁺ flux at any potential. However, large, slowly activating outward cesium currents were observed at all potentials greater than + 20 mV. The difference in skeletal and cardiac Ca²⁺ channel activation kinetics was conserved for outward currents through CEIIIK and SkEIIIK, even at very depolarized potentials (at +100 mV; SkEIIIK: τ_act = 30.7 ± 1.9 ms, n = 11; CEIIIK: τ_act = 2.9 ± 0.5 ms, n = 7). Expression of SkEIIIK in dysgenic myotubes restored both evoked contractions and depolarization-dependent intracellular Ca²⁺ transients with parameters of voltage dependence (V_0.5 = 6.5 ± 3.2 mV and k = 9.3 ± 0.7 mV, n = 5) similar to those for the wild-type DHPR (Garcia, J., T. Tanabe, and K.G. Beam. 1994. J. Gen. Physiol. 103:125–147). However, CEIIIK-expressing myotubes never contracted and failed to exhibit depolarization-dependent intracellular Ca²⁺ transients at any potential. Thus, high Ca²⁺ permeation is required for cardiac-type excitation–contraction coupling reconstituted in dysgenic myotubes, but not skeletal-type. The strong rectification of the EIIIK channels made it possible to obtain measurements of gating currents upon repolarization to −50 mV (Q_off) following either brief (20 ms) or long (200 ms) depolarizing pulses to various test potentials. For SkEIIIK, and not CEIIK, Q_off was significantly (P < 0.001) larger after longer depolarizations to +60 mV (121.4 ± 2.0%, n = 6). The increase in Q_off for long depolarizations exhibited a voltage dependence similar to that of channel activation. Thus, the increase in Q_off may reflect a voltage sensor movement required for activation of L-type Ca²⁺ current and suggests that most DHPRs in skeletal muscle undergo this voltage-dependent transition.