Control Mechanisms of Ca-induced Ca Release

描述（由申请人提供）：在可兴奋的细胞中，通过表面膜的少量Ca2+内流可能激活内质网或肌浆网（SR）上称为ryanodine受体（RyRs）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。这里的例子就是心肌。在这些细胞中，表面去极化激活l型Ca2+通道，产生小的Ca2+通量，激活肌浆网（SR）上的2型ryanodine受体（RyR2）。激活多个RyR2通道在离散位点上的SR产生局部Ca2+释放事件称为火花。在细胞中，火花是ryr2介导的Ca2+释放的基本单位。许多火花的招募和汇总产生驱动心脏收缩的全球Ca2+释放现象。控制ryr2介导的火花的局部控制机制尚不清楚。其中一种机制是局部RyR2 Ca2+激活，通常被称为Ca2+诱导的Ca2+释放（CICR）。CICR是一种直观的自我强化过程，其“爆炸性”的正反馈（即释放的Ca2+应该触发进一步的释放）最终应该清空SR Ca2+存储。这在细胞中不会发生。相反，CICR是被精确控制的，这表明一定存在一些负反馈机制来抵消CICR固有的正反馈。两种细胞质机制，Ca2+依赖性失活和Ca2+依赖性适应，被认为是稳定的负反馈。也有人提出，所需的负控制可能来自sr内局部[Ca2+]变化对RyR2的调节。描述支配RyR2局部Ca2+控制的机制对于理解Ca2+火花的起源显然是必不可少的。这是我们这里的重点，下面的假设将被检验。单个RyR2通道由多种形式的细胞质Ca2+反馈驱动（例如，馈入，邻居诱导和配对脉冲促进）。这是由管腔负控制机制（例如，Ca2+通量减少，直接和间接Ca2+失活）的组合来控制/抵消的，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+释放通道功能的细胞质局部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+释放通道的操作，并最终确定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.