REGULATION OF MEMBRANE EXCITABILITY

这个项目的长期目标是了解分子 潜在的功能、亚细胞定位和 钙通道的生物合成及对钙通道的进一步认识 骨骼肌的兴奋-收缩偶联。在……期间 申请资金，正常肌管和异常肌管表达 将对基因工程钙通道进行研究。具体目标 1.建立二氢吡啶的改进生物物理模型 受体(DHPR)作为L型钙通道和电压的作用 控制SR钙释放的传感器。激活过程 将通过比较慢(骨骼)和 心脏快速激活L型钙通道在发育不良中的表达 肌管。将确定强烈的去极化是否会导致 骨骼L式的经络进入了长寿的开放状态。2.识别 DHPR作为钙通道功能的关键区域，如 作为电荷运动的发生器，也可作为E-C耦合的电压传感器。在……里面 每重复一次，就会进行突变，以扰乱电压依赖性 并因此确定每个重复在生理学中的关键作用 DHPR的职能。骨骼和心脏DHPR的嵌合体将是 被构建为识别对失活重要的区域。其影响 将分析导致低血钾性周期性瘫痪的DHPR突变 从生物物理学上讲。3.探讨DHPR与DHPR相互作用的本质 肌浆网钙释放过程。骨骼/心脏DHPR的构建 嵌合体将被用来识别II-III环中的最小区域 对于骨架型E-C联轴器来说至关重要。相应的寡肽 将被测试以确定它是作为一种激动剂还是拮抗剂 E-C耦合。DHPR激活肌浆网钙释放的化学计量 将会被分析。4.界定不同的形态排列 多种钙通道在肌肉中外源性表达。这个 不同类型钙通道的形态安排将是 用荧光显微镜和电子显微镜表征的 冰冻骨折。5.评估发展的功能后果 骨骼DHPR的变化。DHP的生物合成与组装 肌肉中的受体将被描述出来。生理学意义 骨骼DHPRα1中的可选剪接体的数量将由 目的基因的表达及功能分析。

The long-term goal of this project is to understand the molecular principles underlying the function, subcellular localization and biosynthesis of calcium channels and to further the understanding of excitation-contraction coupling in skeletal muscle. During the period of requested funding, normal myotubes and dysgenic myotubes expressing genetically engineered calcium channels will be studied. The specific aims are to: 1. Produce an improved biophysical model of the dihydropyridine receptor (DHPR) functioning as an L-type calcium channel and as a voltage sensor controlling calcium release from the SR. The activation process will be probed by means of comparisons between slowly (skeletal) and rapidly (cardiac) activating L-type calcium channels expressed in dysgenic myotubes. It will be determined whether the strong depolarization causes the skeletal L-type channel to enter a long-lived open state. 2. Identify regions of the DHPR critical for its function as a calcium channel, as a generator of charge movement and as a voltage sensor for E-C coupling. In each repeat, mutations will be made in order to perturb voltage dependence and, thus, identify the critical roles of each repeat in the physiological functions of the DHPR. Chimeras of the skeletal and cardiac DHPRs will be constructed to identify regions important for inactivation. The effects of DHPR mutations causing hypokalemic periodic paralysis will be analyzed biophysically. 3. Probe the nature of the interaction between the DHPR and the SR calcium release process. Construction of skeletal/cardiac DHPR chimeras will be used to identify a minimal region within the II-III loop critical for skeletal-type E-C coupling. A corresponding oligopeptide will be tested to determine whether it acts as an agonist or antagonist of E-C coupling. The stoichiometry of DHPR activation of SR calcium release will be analyzed. 4. Define the morphological arrangement of different kinds of calcium channels exogenously expressed in muscle. The morphological arrangement of different kinds of calcium channels will be characterized using fluorescence microscopy and using electron microscopy of freeze-fractures. 5. Evaluate functional consequences of developmental changes in the skeletal DHPR. The biosynthesis and assembly of DHP receptors in muscle will be characterized. The physiological significance of alternative splices in the skeletal DHPR alpha1 will be addressed by expression of cDNA and functional analysis.