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

在心肌细胞中，肌浆网(SR)中RyR的钙释放是通过钙诱导-钙释放(CICR)机制激活的。CICR凭借其固有的积极反馈，预计将以一种要么全有要么全不的方式运作。为了产生具有渐变幅度和稳健稳定性的钙瞬变，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RyRs的失活、适应和随机关闭，但尚未有确凿证据。我们最近的研究表明，FK506结合蛋白(FK506-Binding Protein，FKBP)是RyR的一种免疫亲和素和辅助蛋白，通过缩短基本释放事件(钙火花)的持续时间，加速RyR对钙的脱敏，构成CICR的重要调节因子。然而，CICR的主要终止机制仍然难以捉摸。在本研究中，我们用一种新的荧光技术探讨了L类钙通道触发的钙释放终止过程。结合快速、线性的钙指示剂Oregon Green BAPTA 5N和高浓度的钙离子螯合剂EGTA，钙离子的释放被显示为限制在小管-SR连接的离散的钙尖峰，每个尖峰都由单个或几个钙火花组成。在0 mV时，在电压钳脉冲开始后40ms内出现并终止了钙尖峰。用钙通道激动剂FPL64176延长钙通道的开放时间和促进钙通道的重新开放不会延长或触发二次钙峰，即使有2/3的肌质网钙离子仍可被咖啡因释放。潜伏期分析显示，钙尖峰与L类钙通道的首次开放一致，但与重新开放无关。此外，在最初的最大释放后(例如，在0 mV)，即使超极化步骤产生的单位钙电流增加数倍至-120 mV，也未能触发额外的释放，这表明RyRs绝对不稳定。当释放是次极值时(如+30 mV)，超极化时的尾电流确实激活了额外的钙尖峰；共聚焦图像显示它们来自不同的RyR，即去极化时未激发的RyR。这些结果表明，钙释放主要通过RyRs的高度局部化、使用依赖性失活来终止，而不是通过RyRs的随机关闭和适应或耗尽完整的心室肌细胞中的SR Ca~(2+)来终止。最近，我们测量了RyRs从失活状态恢复的时间进程。使用双脉冲方案，我们首先在有FPL存在的情况下逐步将RyRs最大限度地灭活到0 mV，持续50 ms，然后以50~2000 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的恢复服从指数过程，其时间常数约为600ms。有趣的是，异丙肾上腺素对β-肾上腺素能的刺激，在增强RyR激活的同时，并不影响RyR失活的恢复程度和时间进程。这表明激活和失活是RyR的不同性质，服从重叠但不相同的生理调节。-Ryanodine受体；局部钙信号；兴奋-收缩偶联；共聚焦显微镜；心肌细胞

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