Membrane depolarization increases ryanodine sensitivity to Ca 2+ release to the cytosol in L6 skeletal muscle cells: Implications for excitation–contraction coupling

Abstract

The dihydropyridine receptor in the plasma membrane and the ryanodine receptor in the sarcoplasmic reticulum are known to physically interact in the process of excitation–contraction coupling. However, the mechanism for subsequent Ca²⁺ release through the ryanodine receptor is unknown. Our lab has previously presented evidence that the dihydropyridine receptor and ryanodine receptor combine as a channel for the entry of Ca²⁺ under resting conditions, known as store operated calcium entry. Here, we provide evidence that depolarization during excitation–contraction coupling causes the dihydropyridine receptor to disengage from the ryanodine receptor. The newly freed ryanodine receptor can then transport Ca²⁺ from the sarcoplasmic reticulum to the cytosol. Experimentally, this should more greatly expose the ryanodine receptor to exogenous ryanodine. To examine this hypothesis, we titrated L6 skeletal muscle cells with ryanodine in resting and excited (depolarized) states. When L6 muscle cells were depolarized with high potassium or exposed to the dihydropyridine receptor agonist BAYK-8644, known to induce dihydropyridine receptor movement within the membrane, ryanodine sensitivity was enhanced. However, ryanodine sensitivity was unaffected when Ca²⁺ was elevated without depolarization by the ryanodine receptor agonist chloromethylcresol, or by increasing Ca²⁺ concentration in the media. Ca²⁺ entry currents (from the extracellular space) during excitation were strongly inhibited by ryanodine, but Ca²⁺ entry currents in the resting state were not. We conclude that excitation releases the ryanodine receptor from occlusion by the dihydropyridine receptor, enabling Ca²⁺ release from the ryanodine receptor to the cytosol.