Regulation of dynamic behavior of cardiac ryanodine receptor by Mg2+ under simulated physiological conditions

Mg2+, an important constituent of the intracellular milieu in cardiac myocytes, is known to inhibit ryanodine receptor (RyR) Ca2+ release channels by competing with Ca2+ at the cytosolic activation sites of the channel. However, the significance of this competition for local, dynamic Ca2+-signaling processes thought to govern cardiac excitation-contraction (EC) coupling remains largely unknown. In the present study, Ca2+ stimuli of different waveforms (i.e., sustained and brief) were generated by photolysis of the caged Ca2+ compound nitrophenyl (NP)-EGTA. The evoked RyR activity was measured in planar lipid bilayers in the presence of 0.6-1.3 mM free Mg2+ at the background of 3 mM total ATP in the presence or absence of 1 mM luminal Ca2+. Mg2+ dramatically slowed the rate of activation of RyRs in response to sustained (> or =10-ms) elevations in Ca2+ concentration. Paradoxically, Mg2+ had no measurable impact on the kinetics of the RyR response induced by physiologically relevant, brief (<1-ms) Ca2+ stimuli. Instead, the changes in activation rate observed with sustained stimuli were translated into a drastic reduction in the probability of responses. Luminal Ca2+ did not affect the peak open probability or the probability of responses to brief Ca2+ signals; however, it slowed the transition to steady state and increased the steady-state open probability of the channel. Our results indicate that Mg2+ is a critical physiological determinant of the dynamic behavior of the RyR channel, which is expected to profoundly influence the fidelity of coupling between L-type Ca2+ channels and RyRs in heart cells.