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

通过心脏中兰尼定受体 (RYR) 的局部失活来终止 CA2 释放

• 批准号: 6288761
• 负责人: HEPING (PEACE CHENG
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6288761
• 关键词: caffeine; calcium channel; calcium flux; cardiovascular pharmacology; computer simulation; heart contraction; heart electrical activity; laboratory rat; receptor coupling; ryanodine; sarcoplasmic reticulum; thapsigargin

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

在心肌细胞中，肌浆网（SR）中RyR的Ca 2+释放通过Ca 2+诱导的Ca 2+释放（CICR）机制激活。CICR具有固有的积极反馈，预计将以全有或全无的方式运作。为了产生梯度振幅和鲁棒稳定性的Ca 2+瞬变，必须存在调节机制来抵消再生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。FK 506-binding protein（FKBP）是RyR的一种亲免蛋白和辅助蛋白，它通过缩短初级释放事件（Ca ~（2+）火花）的持续时间和加速RyR对Ca ~（2+）的脱敏作用而成为CICR的重要调节因子。然而，CICR的主要终止机制仍不清楚。在本研究中，我们探讨了终止过程的钙释放L-型钙通道触发使用一种新的荧光技术。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，观察到Ca 2+释放为局限于T小管-SR连接处的离散Ca 2+峰，每个峰由单个或几个Ca 2+火花组成。在0 mV时，电压钳脉冲开始后40 ms内出现并终止Ca 2+尖峰。用钙通道激动剂FPL 64176增加钙通道的开放时间和促进钙通道的重新开放，没有延长或触发继发性钙峰，即使2/3的SR钙仍然可用于咖啡因的释放。潜伏期分析表明，钙尖峰与第一次开放，但不与重新开放，L-型钙通道。此外，在初始最大释放（例如，在0 mV），甚至由超极化步骤产生的单位Ca 2+电流的多倍增加至-120 mV也未能触发额外的释放，表明RyR的绝对不应性。当释放为次最大时（例如，在+30 mV），超极化后的尾电流确实激活了额外的Ca 2+尖峰;共聚焦图像显示它们起源于不同的RyR，即，在去极化过程中未被激发的。这些结果表明，Ca 2+释放终止主要是由一个高度本地化的，使用依赖性的RyRs失活，但不是随机关闭和适应RyRs或耗尽SR Ca 2+在完整的心室肌细胞。最近，我们测量了RyR从失活中恢复的时间过程。使用双脉冲方案，我们首先在存在FPL的情况下通过步进至0 mV持续50 ms最大程度地灭活RyR，然后以50至2000 ms的间隔递送0 mV的测试脉冲。我们的结果表明，RyR恢复遵循指数过程的时间常数约为600毫秒，在我们的实验条件下。有趣的是，β-肾上腺素能刺激异丙肾上腺素，而增强RyR激活，并没有影响RyR失活的恢复的程度和时间过程。这表明激活和失活是RyR的不同性质，受到重叠但不相同的生理调节。- 兰尼碱受体;局部钙信号;兴奋-收缩偶联;共聚焦显微镜;心肌细胞