Control Mechanisms of Ca-induced Ca Release

描述(申请人提供)：在可兴奋的细胞中，通过表面膜的少量钙内流可能激活内质或肌质网(SR)上称为兰诺定受体(RyRs)的细胞内钙释放通道。由此产生的RyR介导的钙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，心肌就是一个很好的例子。在这些细胞中，表面去极化激活了L类型的钙通道，产生了一个小的钙通量，激活了肌浆网(SR)上的2型兰诺定受体(RyR2)。在SR上的离散位置激活多个RyR2通道会产生称为火花的局部钙释放事件。在细胞中，火花是RyR2介导的钙释放的基本单位。许多火花的募集和总和产生了推动心肌收缩的全球钙释放现象。对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钙激活，通常被称为钙诱导钙释放(CICR)。CICR在直觉上是一个自我强化的过程，它的“爆炸性”正反馈(即，释放的钙离子应该会触发进一步的释放)最终应该会清空SR的钙离子储存库。这在细胞中是不会发生的。相反，CICR受到精确控制，这表明必须存在某种负反馈机制(S)来对抗CICR固有的正反馈。有两种胞浆机制，即钙依赖的失活和钙依赖的适应，被认为是稳定的负反馈。也有人提出，所需的负调控可能来自SR内部局部[Ca~(2+)]变化对RyR2的调控。阐明控制RyR2局部钙调控的机制显然对于理解钙火花的来源是至关重要的。这是我们在这里的重点，下面的假设将得到检验。单个RyR2通道由多种形式的胞浆钙反馈(例如，馈通、邻居诱导和成对脉冲促进)驱动。这是由管腔负性控制机制(例如，钙通量减少、直接和间接钙失活)的组合来控制/对抗的，以最终定义钙火花的时空性质。具体目标是：具体目标1：定义胞浆局部钙正反馈，驱动单个RyR2钙释放通道的功能。具体目标#2：明确管腔内钙负调控机制，该机制支配单个RyR2钙释放通道的运行，并最终控制钙火花的时空性质。

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.