Control Mechanisms of Ca-induced Ca Release

描述（由申请人提供）：在可兴奋细胞中，通过表面膜的少量 Ca2 流入可能会激活内质网或肌浆网 (SR) 上称为兰尼碱受体 (RyR) 的细胞内 Ca2 释放通道。由此产生的 RyR 介导的 Ca2 释放调节许多细胞过程，如收缩、分泌、突触传递、受精、核孔调节和转录。这里，典型的例子是心肌。在这些细胞中，表面去极化激活 L 型 Ca2 通道，产生少量 Ca2 通量，从而激活肌浆网 (SR) 上的 2 型兰尼碱受体 (RyR2)。 SR 上离散位点的多个 RyR2 通道的激活会产生称为火花的局部 Ca2 释放事件。在细胞中，火花是 RyR2 介导的 Ca2 释放的基本单位。许多火花的募集和求和产生了驱动心肌收缩力的全局 Ca2 释放现象。人们对控制 RyR2 介导的火花的局部控制机制知之甚少。这些机制之一是局部 RyR2 Ca2 激活，通常称为 Ca2 诱导的 Ca2 释放 (CICR)。 CICR 直观上是一个自我强化过程，其“爆炸性”正反馈（即释放的 Ca2 应触发进一步释放）最终应清空 SR Ca2 存储。这不会发生在细胞中。相反，CICR 受到精确控制，表明必须存在一些负反馈机制来抵消 CICR 固有的正反馈。两种胞质机制，Ca2 依赖性失活和 Ca2 依赖性适应，已被认为是稳定负反馈。也有人提出，所需的负控制可能来自于 SR 内局部 [Ca2+] 变化对 RyR2 的调节。描述 RyR2 局部 Ca2 控制的机制对于理解 Ca2 火花的起源显然至关重要。这是我们这里的重点，下面的假设将得到检验。单个 RyR2 通道由多种形式的胞质 Ca2+ 反馈驱动（例如，馈通、邻近诱导和配对脉冲促进）。这是通过管腔负控制机制（例如 Ca2 通量减少、直接和间接 Ca2 失活）的组合来控制/抵消的，以最终定义 Ca2 火花的时空性质。具体目标是： 具体目标#1：定义驱动单个 RyR2 Ca2 释放通道功能的胞质局部 Ca2 正反馈。具体目标#2：定义管腔 Ca2 负控制机制，控制单个 RyR2 Ca2 释放通道的运行，并最终控制 Ca2 火花的时空性质。

DESCRIPTION (provided by applicant): In excitable cells, a small Ca2+ influx through the surface membrane may activate intracellular Ca2+ release channels called ryanodine receptors (RyRs) on the endoplasmic or sarcoplasmic reticulum (SR). The resulting RyR-mediated Ca2+ release regulates many cellular processes like contraction, secretion, synaptic transmission, fertilization, nuclear pore regulation and transcription. Here, the case in point is cardiac muscle. In these cells, surface depolarization activates an L-type Ca2+ channel generating a small Ca2+ flux which activates type-2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Activation of multiple RyR2 channels at discrete sites on the SR generates localized Ca2+ release events called sparks. In cells, sparks are the elemental unit of RyR2-mediated Ca2+ release. Recruitment and summation of many sparks generates the global Ca2+ release phenomena that drive cardiac contractility. The local control mechanisms that govern the RyR2-mediated spark are poorly understood. One of these mechanisms is local RyR2 Ca2+ activation, often referred to as Ca2+-induced Ca2+ release (CICR). CICR is an intuitively a self-reinforcing process whose "explosive" positive feedback (i.e., released Ca2+ should trigger further release) should ultimately empty the SR Ca2+ store. This does not happen in cells. Instead, CICR is precisely controlled indicating that some negative-feedback mechanism(s) must exist to counter the inherent positive feedback of CICR. Two cytosolic mechanisms, Ca2+-dependent inactivation and Ca2+-dependent adaptation, have been proposed to be the stabilizing negative feedback. It has also been proposed that the needed negative control may arise from RyR2 regulation by local [Ca2+] changes inside the SR. Delineating mechanisms that govern RyR2 local Ca2+ control is clearly essential to understanding the origin of the Ca2+ spark. This is our focus here and the following hypothesis will be tested. Single RyR2 channels are driven by multiple forms of cytosolic Ca2+ feedback (e.g., feed through, neighbor-induced & paired pulse facilitation). This is controlled/countered by a combination of lumenal negative control mechanisms (e.g., Ca2+-flux reduction, direct & indirect Ca2+ deactivation) to ultimately define the spatiotemporal nature of the Ca2+ spark. The specific aims are: Specific Aim #1: Define the cytosolic local Ca2+ positive feedback that drives the function of single RyR2 Ca2+ release channels. Specific Aim #2: Define lumenal Ca2+ negative control mechanisms that govern operation of single RyR2 Ca2+ release channels and ultimately the spatiotemporal nature of the Ca2+ spark.