TERMINATE CA2+ RELEASE BY LOCAL INACTIVATION OF RYANODINE RECEPTORS(RYR)IN HEART

在心肌细胞中，肌浆网（SR）中RyR的Ca2+释放是由Ca2+诱导的Ca2+释放（CICR）机制激活的。CICR由于其固有的正反馈，预计将以全有或全无的方式运作。为了产生渐变振幅和稳健稳定性的Ca2+瞬态，必须存在一种调节机制来抵消再生的CICR。已经提出了几种机制，包括ryr的失活、适应和随机关闭，但尚未有确凿的证据记录。我们最近的研究表明，fk506结合蛋白（FKBP）是一种RyR的亲免疫蛋白和辅助蛋白，通过缩短基本释放事件（Ca2+火花）的持续时间和加速RyR对Ca2+的脱敏，构成了CICR的重要调节剂。然而，CICR的主要终止机制尚不清楚。在本研究中，我们利用一种新的荧光技术探测了l型Ca2+通道触发的Ca2+释放的终止过程。通过结合快速，线性Ca2+指示剂，俄勒冈绿BAPTA 5N，和高浓度的Ca2+螯合剂，EGTA， Ca2+释放被可视化为离散的Ca2+尖峰限制在t小管- sr连接，每个由单个或几个Ca2+火花组成。在0 mV时，Ca2+尖峰发生并在电压箝位脉冲开始后的40 ms内终止。使用Ca2+通道激动剂FPL64176增加Ca2+通道的开放时间和促进Ca2+通道的重新开放，并没有延长或触发二次Ca2+峰值，即使2/3的SR Ca2+仍然可以通过咖啡因释放。潜伏期分析表明，Ca2+峰值与l型Ca2+通道的首次开放一致，但与再开放不一致。此外，在初始最大释放（例如，在0 mV）之后，即使通过超极化步骤产生的单一Ca2+电流增加数倍至-120 mV也未能触发额外的释放，这表明ryr的绝对耐火性。当释放为次极大时（例如，在+30 mV），超极化后的尾电流确实激活了额外的Ca2+峰；共聚焦图像显示它们来自不同的ryr，即在去极化期间未发射的ryr。这些结果表明，Ca2+释放主要是由高度局部的、使用依赖性的RyRs失活终止的，而不是由RyRs的随机关闭和适应或完整心室肌细胞中SR Ca2+的消耗终止的。最近，我们测量了ryr从失活中恢复的时间过程。使用双脉冲协议，我们首先在FPL存在的情况下将ryr逐步灭活至0 mV，持续50 ms，然后以50至2000 ms的间隔提供0 mV的测试脉冲。结果表明，在我们的实验条件下，RyR恢复遵循指数过程，时间常数为~600 ms。有趣的是，异丙肾上腺素刺激β -肾上腺素能增强RyR的激活，但不影响RyR失活的恢复程度和时间过程。这表明RyR的激活和失活是不同的特性，受到重叠但不相同的生理调节。-赖诺定受体；局部钙信号；兴奋收缩耦合;共焦显微镜;心脏细胞

SUMMARY OF WORKIn 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. More recently we measured the time course of recovery of RyRs from inactivation. Using double-pulse protocols, we first maximally inactivated the RyRs by step to 0 mV for 50 ms in the presence of FPL, and then, delivered a test pulse of 0 mV at 50 to 2000 ms intervals. Our results indicate that RyR recovery follows an exponential process with a time constant of ~600 ms under our expeimental conditions. Interstingly, beta-adrenergic stimulation by isopreterenol, while enhancing RyR activation, did not affect the extent and time course of recovery of RyR inactivation. This suggests that activation and inactivation are different properties of RyR, subjecting to overlapping but not identical physiological regulations. - Ryanodine receptors; Local calcium signaling; Excitation-contraction coupling; Confocal microscopy; Cardiac myocytes