RECONSTITUTION OF INTRACELLULAR CA2+ CHANNELS

兰尼定和肌醇等细胞内钙库的钙通道 1，4，5三磷酸(IP3)受体，最终负责 观察电压刺激下细胞内钙离子的变化， 神经递质和荷尔蒙。活化动力学和反应动力学 细胞内钙通道的失活也可能是 对已知的普遍存在的细胞内钙释放机制负责 作为钙离子诱导的钙离子释放(CICR)。该提案的重点是 心脏兰尼定受体和IP3受体的分子特性 和骨骼肌细胞有兴趣了解 这些通道对兴奋-收缩(EC)耦合的贡献。 该提案的目的是研究1)钙离子的动力学- 兰尼定受体的依赖激活和可能的失活。 这将建立兰诺定受体的离子条件 可能介导细胞内的CICR；2)钙结合部位的缺陷 兰尼定受体在猪恶性高热模型中的作用 一种影响兰尼定受体的遗传性疾病。这是一个很有希望的 模型可以帮助我们理解结构和动力学基础 依赖于钙离子的激活和失活；3)钙离子池的大小 受Ryanodine和IP3控制及其介导的钙释放速率 心肌和骨骼肌中的受体。这一点很重要，因为 这两种通道均可增加心肌细胞的钙离子通透性。 肌浆网；4)兰尼定的阻断或激活 由蝎子毒素引起的受体。这将确定是否可以使用毒素 分析Ryanodine受体在EC偶联中的作用。这个 四个目标是从以前的资助中获得的经验中得出的 细胞内钙离子通道功能重建期 和表面来源，使用45Ca2+通量、蛋白质的组合 纯化和平面双层记录。这项提议的力度 取决于这些技术在 数量框架。这些实验对于理解 细胞内钙离子通道的功能特性及其动力学 细胞内环境对这些通道施加的限制。 该提案应提供有关下列机制的新资料 横纹肌细胞内钙释放的实验研究 并对有关钙离子释放的新假说进行了严格的检验 由钙离子和IP3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.