CARDIAC CA CHANNELS--REGULATION OF SLOW GATING BEHAVIOR

调节控制打开和关闭的过程，或 门控，钙通道是至关重要的控制， 心跳关于如何监管渠道的经典观点集中在 在几乎总是 可在短时间内开放。相比之下，最近的研究表明， 暗示了一种新的可能性， 调节钙通道是缓慢地在两个根本性的 不同的门控模式;（1）主动模式，其中通道开口 是可能的，和（2）一个“休眠”模式，其中通道是不可能的 打开.本项目的总体目标是利用膜片钳技术 技术，以建立一个严格的理解，在单一的渠道 和分子水平，钙通道之间的缓慢转换 心脏中的活动和休眠门控模式。 将通过贴片测量单一的“L型”Ca通道电流 单个哺乳动物心室细胞的电压钳技术。一个新 分析方法，称为“扫描直方图分析”，将应用于 提供定量证据，证明不同的 活动和休眠门控模式，通道在这两种模式之间缓慢循环。 该方法还将使明确的动力学识别 模型来解释门控模式之间的转换。的验证 这样的模型开启了新的区分特定 受调节Ca通道的因素影响的动力学步骤。 这些因子包括通道磷酸化的调节剂（β-肾上腺素能 和胆碱能激动剂）或去磷酸化（冈田酸），以及 与通道直接相互作用的试剂（膜电压， 合成的Ca通道配体和G蛋白）。扫描直方图比较 这些因素对细胞内应用的作用的影响 蛋白酶将提供重要的线索，以存在域上 对减缓门控转换至关重要的通道分子 模式之间。 阐明了使Ca通道偏向活性或 休眠门控模式承诺提供基本的洞察力， 钙通道调控的分子机制。

Regulation of the processes that control the opening and closing, or gating, of Ca channels is crucially important to the control of the heartbeat. Classical ideas about how channels are regulated have focused upon changes in the gating behavior of channels that are almost always available to open upon short notice. In contrast, recent studies have hinted at the novel possibility that a predominant mechanism for modulating Ca channels is to shift channels slowly between two radically different modes of gating; (1) an active' mode in which channel openings are probable, and (2) a 'hibernating'mode in which channels are unlikely to open. The overall goal of this project is to use patch clamp techniques to establish a rigorous understanding, at the single channel and molecular level, of the slow transitions of the Ca channel between active and hibernating gating modes in the heart. Unitary "L-type" Ca channel currents will be measured by the patch voltage clamp technique in single mammalian ventricular cells. A new analytic approach, termed "sweep histogram analysis," will be applied to provide quantitative evidence for the genuine existence of distinct active and hibernating gating modes, between which channels cycle slowly. The approach will also enable the identification of explicit kinetic models to explain the transitions between gating modes. The validation of such a model opens the possibility to newly distinguish the specific kinetic steps that are affected by factors that modulate the Ca channel. These factors include regulators of channel phosphorylation (B-adrenergic and cholinergic agonists) or dephosphorylation (okadaic acid), as well as agents that interact with the channel directly (membrane voltage, synthetic Ca channel ligands, and G proteins). Sweep histogram comparison of the effects of these factors to the action of intracellularly applied proteases will provide important clues as to the existence of domains on the channel molecule that may be crucial to slow gating transitions between modes. Clarification of the mechanisms that bias Ca channels toward active or hibernating gating modes promises to provide fundamental insight into the molecular mechanisms by which the Ca channel is regulated.