Terminate CA2+ Release by Local Inactivation of Ryanodine Receptors(RyR)in Heart

在心肌细胞中，Ca 2 + 肌浆网（SR）中RyR的释放被激活， Ca 2+诱导的Ca 2+释放（CICR）机制。CICR，其 固有的正反馈，预计将在一个 “全有或全无”的时尚。为了产生Ca 2+瞬变， 梯度振幅和强大的稳定性，监管机制必须 存在以抵消再生CICR。几种机制， 包括RyR的失活、适应和随机关闭 已经提出，但还没有确凿的证据， 记录在案。我们最近的研究表明，FK 506结合 蛋白（FKBP），一种亲免蛋白和RyR的辅助蛋白， 构成了一个突出的调节CICR通过缩短 基本释放事件（Ca 2+火花）的持续时间，以及 促进RyR对Ca ~（2+）的脱敏。但 CICR的主要终止机制仍不清楚。在 本研究探讨了Ca 2+释放的终止过程 用一种新的荧光探针， 法通过结合快速、线性的Ca 2+指示剂，俄勒冈州 绿色BAPTA 5 N和高浓度的Ca 2+螯合剂， EGTA，Ca 2+释放被可视化为离散的“Ca 2+尖峰”。 仅限于T管-SR连接，每个连接由单个或一个 少量Ca 2+火花。在0 mV时，出现并终止Ca 2+峰电位 在电压箝位脉冲开始后的40 ms内。 增加开放时间，促进Ca 2+再开放 Ca 2+通道激动剂FPL 64176的钙通道， 延长或触发继发性Ca 2+尖峰，即使2/3的 SR Ca 2+仍然可以通过咖啡因释放。延迟 分析显示Ca 2+尖峰与第一次开口一致， 但不与L-型Ca 2+通道的重新开放有关。此外，委员会认为， 在初始最大释放（例如，0 mV），甚至是多倍 由超极化产生的单位Ca 2+电流增加 阶跃至-120 mV未能触发额外释放，表明 RyR的绝对不应期。当释放为次最大时 (e.g.,在+30 mV时），超极化后的尾电流确实激活 额外的Ca 2+尖峰;共聚焦图像显示， 源自不同的RyR，即，那些未被解雇的人 去极化这些结果表明，Ca 2+释放是 主要由高度本地化的、依赖于使用的 RyR的失活，但不是通过随机关闭和适应 RyRs或SR Ca ~（2+）耗竭。

SUMMARY OF WORK In cardiac myocytes, Ca2+ release from RyR in the sarcoplasmic reticulum (SR) is activated by the Ca2+-induced-Ca2+ release (CICR) mechanism. CICR, with its inherent positive feedback, is expected to operate in an "all-or-none" fashion. In order to generate Ca2+ transients of graded amplitude and robust stability, a regulatory mechanism must exist to counteract the regenerative CICR. Several mechanisms, including inactivation, adaptation, and stochastic closing of RyRs have been proposed, but no conclusive evidence has yet been documented. Our recent study has shown that FK506-binding protein (FKBP), an immunophilin and accessory protein of RyR, constitutes a prominent regulator of CICR via shortening the duration of the elementary release events (Ca2+ sparks) and accelerating the desensitization of RyR to Ca2+. However, the primary termination mechanism of CICR remained elusive. In the present study, we probed the termination process of Ca2+ release triggered by L-type Ca2+ channel using a novel fluorescent technique. By combination of a fast, linear Ca2+ indicator, Oregon Green BAPTA 5N, and a high concentration of Ca2+ chelator, EGTA, Ca2+ release was visualized as discrete "Ca2+ spikes" restricted toT tubule-SR junctions, each consisting of single or a few Ca2+ sparks. At 0 mV, Ca2+ spikes occurred and terminated within 40 ms following the onset of voltage clamp pulses. Increasing the open duration and promoting the reopenings of Ca2+ channels with the Ca2+ channel agonists, FPL64176, did not prolong or trigger secondary Ca2+ spikes, even though 2/3 of the SR Ca2+ remained available for release by caffeine. Latency analysis revealed that Ca2+ spikes coincided with the first openings, but not with the reopenings, of L-type Ca2+ channels. Furthermore, after an initial maximal release (e.g., at 0 mV), even a multi-fold increase in unitary Ca2+ current produced by a hyperpolarization step to -120 mV failed to trigger additional release, indicating an absolute refractoriness of RyRs. When the release was submaximal (e.g., at +30 mV), tail currents upon hyperpolarization did activate additional Ca2+ spikes; confocal images revealed that they originated from a different RyRs, i.e., those unfired during depolarization. These results indicate that Ca2+ release is terminated primarily by a highly localized, use-dependent inactivation of RyRs , but not by stochastic closing and adaptation of RyRs or depletion of SR Ca2+ in intact ventricular myocytes.