RECONSTITUTION OF INTRACELLULAR CALCIUM CHANNELS

细胞内钙库的钙通道，如ryanodine和肌醇 1，4，5三磷酸盐（IP 3）受体，最终负责 在电压刺激的细胞中观察到的细胞溶质Ca 2+的变化， 神经递质和激素。 活化动力学和 细胞内Ca 2+通道的失活也可能是 负责已知的普遍存在的细胞内Ca 2+释放机制 Ca 2+诱导的Ca 2+释放（CICR）。 该提案的重点是 心脏兰尼碱受体和IP 3受体的分子特性 和骨骼肌细胞， 这些通道对兴奋-收缩（EC）偶联的贡献。 该提案的目的是研究1）Ca 2 +- 依赖性激活和可能的灭活兰尼碱受体。 这将建立兰尼碱受体 可能介导细胞中的CICR; 2）Ca 2+结合位点的缺陷， 恶性高热的猪模型中的兰尼碱受体， 一种影响兰尼碱受体的遗传性疾病 这是一个很有前景 模型，可以帮助我们理解的结构和动力学基础， Ca 2+依赖的激活和失活; 3）Ca 2+池的大小 由ryanodine和IP 3控制和介导的Ca 2+释放速率 心脏和骨骼肌中的受体。 这一点很重要，因为 这两种类型的通道可以用来增加Ca 2+的渗透性， 肌浆网;和4）阻断或激活ryanodine 蝎子毒素的受体。 这将确定是否可以使用毒素 分析ryanodine受体对EC偶联的贡献。 的 根据以往资助的经验，提出了四个目标 细胞内Ca 2+通道的功能重建 和表面起源，使用45 Ca 2+通量，蛋白质 纯化和平面双层记录。 提案的力度 存在于这些技术在用于 量化框架。 这些实验对于理解 细胞内Ca ~（2+）通道的功能特性和动力学 细胞内环境对这些通道的限制。 该提案应提供关于下列机制的新资料： 横纹肌细胞内钙库的钙释放 并对有关Ca 2+释放的新假设进行了严格的检验 由Ca 2+和IP 3触发。

Ca2+ channels of intracellular Ca2+ stores, such as ryanodine and inositol 1, 4, 5 trisphosphate (IP3) receptors, are ultimately responsible for the changes in cytosolic Ca2+ observed in cells stimulated by voltage, neurotransmitters, and hormones. The kinetics of activation and inactivation of intracellular Ca2+ channels is likely also to be responsible for the ubiquitous intracellular Ca2+ release mechanism known as Ca2+- induced Ca2+ release (CICR). The proposal focuses on the molecular properties of ryanodine receptors and IP3 receptors of cardiac and skeletal muscle cells with the interest in understanding the contribution of these channels to excitation-contraction (EC) coupling. The objectives of the proposal are to study 1) the kinetics of the Ca2+- dependent activation and possibly, inactivation of ryanodine receptors. This will establish the ionic conditions under which ryanodine receptors may mediate CICR in the cell; 2) the defects in Ca2+ binding sites of ryanodine receptors in the porcine model of Malignant Hyperthermia which is a genetic disorder affecting ryanodine receptors. This is a promising model that may help us to understand the structural and kinetic basis of Ca2+-dependent activation and inactivation; 3) the size of the Ca2+ pools controlled by, and rates of Ca2+ release mediated by, ryanodine and IP3 receptors in cardiac and skeletal muscle. This is important given that both types of channels may serve to increase the Ca2+ permeability of the sarcoplasmic reticulum; and 4) the block or activation of ryanodine receptors by scorpion toxins. This will establish if toxins could be used to dissect the contribution of ryanodine receptors to EC coupling. The four goals are drawn from the experience gained in the previous funding period on the functional reconstitution of Ca2+ channels, of intracellular and surface origin, using a combination of 45Ca2+ fluxes, protein purification, and planar bilayer recording. The strength of the proposal resides in the resolving power of these techniques when used within a quantitative framework. The experiments are essential for understanding the functional properties of intracellular Ca2+ channels and the kinetic restrictions imposed on these channels by the intracellular environment. The proposal should provide new information regarding the mechanisms of Ca2+ release from intracellular stores operative in striated muscle cells and provide rigorous tests of novel hypothesis concerning Ca2+ release triggered by Ca2+ and IP3.