Termination of Ca2+ release by a local inactivation of ryanodine receptorsin cardiac myocytes

In heart, a robust regulatory mechanism is required to counteract the regen erative Ca2+-induced Ca2+ release from the sarcoplasmic reticulum. Several mechanisms, including inactivation, adaptation, and stochastic closing of r yanodine receptors (RyRs) have been proposed, but no conclusive evidence ha s yet been provided. We probed the termination process of Ca2+ release by u sing a technique of imaging local Ca2+ release, or "Ca2+ spikes", at subcel lular sites; and we tracked the kinetics of Ca2+ release triggered by L-typ e 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 p romoting the reopenings of Ca2+ channels with the Ca2+ channel agonist, FPL 64176, did not prolong or trigger secondary Ca2+ spikes, even though two-th irds of the sarcoplasmic reticulum Ca2+ remained available for release. Lat ency of Ca2+ spikes coincided with the first openings but not with the reop enings of L-type Ca2+ channels. After an initial maximal release, even a mu lti-fold increase in unitary Ca2+ current induced by a hyperpolarization to -120 mV failed to trigger additional release, indicating absolute refracto riness of RyRs. When the release was submaximal (e.g., at +30 mV), tail cur rents did activate additional. Ca2+ spikes; confocal images revealed that t hey originated from RyRs unfired during depolarization. These results indic ate that Ca2+ release is terminated primarily by a highly localized, use-de pendent inactivation of RyRs but not by the stochastic closing or adaptatio n of RyRs in intact ventricular myocytes.