MOLECULAR PHYSIOLOGY OF NEUROMUSCULAR DISEASES

许多遗传性肌肉疾病都是由骨骼肌的异常引起的。 肌膜性高钾型周期性麻痹的电兴奋性 (HPP)虚弱的发作与 细胞外钾在发作期间，肌肉去极化， 不带电的 一种相关的疾病，先天性副肌强直 (PMC)，其特征在于局部冷诱导僵硬（肌强直）， 轻度虚弱 肌强直由重复性后放电引起， 起源于受影响的肌肉，独立于神经元输入。 生理学和遗传学证据的结合已经证实， HPP、PMC和马型周期性麻痹都是由 在成人骨骼肌同种型的α亚单位的突变， 钠离子通道我们以前已经证明，主要功能 HPP中的缺陷是Na电流失活的破坏。的损失 人HPP肌管中的失活通过提高细胞外 [K].这就解释了袭击的间歇性， 但在生物物理学上却出乎意料。 这项建议的一个主要目的是 确定细胞外K是否直接改变突变体中的门控 渠道，探讨如何K发挥其影响，并阐明 持续钠电流的动力学基础。Na的门控行为 通道从未在温度敏感表型中测量， PMC。 由PMC突变产生的功能缺陷将由以下定义： 突变cDNA在哺乳动物细胞中的异源表达。许多PMC 突变发生在III-IV细胞质环，和额外的位点- 将进行定向诱变以确定该结构域如何 参与失活过程。 异常钠通道 HPP和PMC中的行为将被合并到计算机中， 模拟和动物模型，以探索病理生理基础， 这些表型的显性表达。在马周期性麻痹中 所有受影响的动物在α亚单位中具有相同的点突变， 苯妥英可以降低发作频率。单位钠电流 将从马肌管记录以定义功能缺陷， 以测量苯妥英对异常通道门控的影响。 拟议的研究旨在全面了解 两种人类神经肌肉疾病的分子生理学基础。在 此外，这些结果将进一步加深我们对Na通道的理解 在分子水平上的功能，将提供见解， 为这些疾病设计合理的治疗方法，并将作为一个模型， 用于理解改变的电生理的其他病症的系统 兴奋性（癫痫、心律失常）。

Many inherited disorders of muscle are caused by an abnormality in the electrical excitability of the sarcolemmal hyperkalemic periodic paralysis (HPP) episodes of weakness occur in association with an elevation in extracellular potassium. During an attack, muscles are depolarized and electrically inexcitable. A related disorder, paramyotonia congenita (PMC), is characterized by localized cold-induced stiffness (myotonia) and mild weakness. Myotonia arises from repetitive after-discharges that originate in affected muscle independent from neuronal input. A combination of physiologic and genetic evidence has established that HPP, PMC and an equine form of periodic paralysis are all caused by mutations in the alpha subunit of the adult skeletal muscle isoform of the sodium channel. We have shown previously that the primary functional defect in HPP is a disruption of Na current inactivation. The loss of inactivation in human HPP myotubes was enhanced by raised extracellular [K]. This provided an explanation for the episodic nature of the attacks, but was unexpected biophysically. A major aim of this proposal is to determine whether extracellular K directly alters gating in mutant channels, to explore how K exerts its influence, and to elucidate the kinetic basis for the persistent Na current. Gating behavior of Na channels has never been measured in the temperature-sensitive phenotype, PMC. The functional defects produced by PMC mutations will be defined by heterologous expression of mutant cDNAs in mammalian cells. Many PMC mutations occur in the III-IV cytoplasmic loop, and additional site- directed mutagenesis will be performed to define how this domain participates in the process of inactivation. The aberrant Na channel behaviors in HPP and PMC will be incorporated into both a computer simulation and an animal model to explore the pathophysiologic basis for the dominant expression of these phenotypes. In equine periodic paralysis all affected animals have the same point mutation in the alpha subunit, and the frequency of attacks is reduced by phenytoin. Unitary Na currents will be recorded from equine myotubes to define the functional defect and to measure the effects of phenytoin on aberrant channel gating. The proposed studies are designed to provide a complete understanding of the molecular physiologic basis of two human neuromuscular diseases. In addition, these results will further our understanding of Na channel function at the molecular level, will provide insights from which to design rational therapy for these diseases, and will serve as a model system for understanding other disorders of altered electrical excitability (epilepsy, cardiac dysrhythmias).