Termination of Ca2+ Release by a Local Inactivation of Ryanodine Receptors in Cardiac Myocytes

In heart, a robust regulatory mechanism is required to counteract the regenerative Ca²⁺-induced Ca²⁺ release from the sarcoplasmic reticulum. Several mechanisms, including inactivation, adaptation, and stochastic closing of ryanodine receptors (RyRs) have been proposed, but no conclusive evidence has yet been provided. We probed the termination process of Ca²⁺ release by using a technique of imaging local Ca²⁺ release, or "Ca²⁺ spikes", at subcellular sites; and we tracked the kinetics of Ca²⁺ release triggered by L-type Ca²⁺ channels. At 0 mV, Ca²⁺ release occurred and terminated within 40 ms after the onset of clamp pulses (0 mV). Increasing the open-duration and promoting the reopenings of Ca²⁺ channels with the Ca²⁺ channel agonist, FPL64176, did not prolong or trigger secondary Ca²⁺ spikes, even though two-thirds of the sarcoplasmic reticulum Ca²⁺ remained available for release. Latency of Ca²⁺ spikes coincided with the first openings but not with the reopenings of L-type Ca²⁺ channels. After an initial maximal release, even a multi-fold increase in unitary Ca²⁺ current induced by a hyperpolarization to -120 mV failed to trigger additional release, indicating absolute refractoriness of RyRs. When the release was submaximal (e.g., at +30 mV), tail currents did activate additional Ca²⁺ spikes; confocal images revealed that they originated from RyRs unfired during depolarization. These results indicate that Ca²⁺ release is terminated primarily by a highly localized, use-dependent inactivation of RyRs but not by the stochastic closing or adaptation of RyRs in intact ventricular myocytes.