Disease Pathogenesis and Modification for CaV1.1-Associated Hypokalemic Periodic

描述(申请人提供)：低血钾性周期性麻痹(HypoPP)是一种主要遗传的骨骼肌疾病，其反复发作的无力是由纤维电兴奋性的间歇性失效引起的。发作发生在与 低钾血症(K+&lt；3 mm)，可由碳水化合物摄入、运动或压力引发。HypoPP的分子缺陷是异质性的，有60%的家系编码L型钙通道CaV1.1的CACNA1有错义突变，另外20%的家系有编码电压门控Na通道NaV1.4的SCN4A错义突变，其余的家系尚未确定。尽管有这样的科学进步，但纤维去极化和兴奋性丧失的瞬时发作的致病基础还没有完全建立起来。奇怪的是，NaV1.4中的所有8个突变和CaV1.1中的6个突变都发生在S4电压敏感区的精氨酸残基上。到目前为止，在切开的卵母细胞中研究的所有6个NaV1.4-HypoPP突变都揭示了一个微小的异常阳离子电流，它通过突变的S4片段和通道蛋白之间的“门控孔”传导，在超极化电位下激活。我们最近报道了NAV1.4-R669H小鼠肌肉纤维中的门控孔电流。这种门控气孔电导被认为是触发低K+中HypoPP纤维反常去极化的内向电流的来源。一个主要的悬而未决的问题是，与HypoPP相关的CaV1.1中的同源R/X突变是否也产生门控孔流，从而为Nav1.1或CaV1.1突变引起的HypoPP的常见病理机制提供支持证据。这个项目的总体目标是更好地了解CaV1.1突变导致的HypoPP的病理基础。我们已经使用基因打靶的方法产生了CaV1.1的R528H敲击突变作为HypoPP的模型。本项目的目的是：(1)扩展CaV1.1-R528H小鼠的低钾性周期性瘫痪的表型特征；(2)检验CaV1.1-R528H通道传导异常门控孔流的假设；(3)表征CaV1.1-R528H肌肉纤维中钙释放的完整性；(4)在CaV1.1-HypoPP小鼠模型中探索潜在的疾病改良剂。这项工作将扩大我们对HypoPP虚弱发作的发病机制的理解，并将提供一个模型系统来测试治疗策略的有效性，作为一种减轻或改善疾病负担的手段，并为所提出的疾病机制提供验证性的实验支持。 与公共卫生相关：家族性周期性麻痹是一种罕见的骨骼肌疾病，肌肉兴奋性的间歇性丧失会导致严重虚弱或持续数小时至数天的瘫痪发作。这种分子缺陷十多年来一直被认为是钙通道或钠通道的突变，但这些通道缺陷导致虚弱发作的易感性的机制尚不清楚，因此缺乏合理的治疗策略来改变病程。我们已经开发出唯一的基于钙通道突变的周期性瘫痪的基因工程小鼠模型，并将使用这一独特的工具进一步定义瘫痪发作的潜在机制，并测试潜在的干预措施以改变疾病负担。

DESCRIPTION (provided by applicant): Hypokalemic periodic paralysis (HypoPP) is a dominantly inherited disorder of skeletal muscle in which recurrent attacks of weakness are caused by intermittent failure of fiber electrical excitability. Episodes occur in association with hypokalemia (K+ < 3 mM) and may be triggered by carbohydrate ingestion, exercise, or stress. The molecular defect in HypoPP is heterogeneous, with 60% of families having missense mutations in CACNA1S encoding the L-type Ca channel CaV1.1, another 20% have missense mutations in SCN4A encoding the voltage-gated Na channel NaV1.4, and the remainder undetermined. Despite this scientific advance, the pathogenic basis for the transient attacks of fiber depolarization with loss of excitability is not fully established. Curiously, all 8 mutationsin NaV1.4 and 6 of 7 in CaV1.1 occur at arginine residues in S4 voltage-sensor domains. Thus far, all 6 NaV1.4-HypoPP mutations studied in the cut-open oocyte have revealed a small anomalous cation current activated at hyperpolarized potentials, via conduction through a "gating pore" between the mutated S4 segment and the channel protein. We recently reported a gating pore current in muscle fibers from NaV1.4-R669H mice. This gating pore conductance is hypothesized to be the source of the inward current that triggers the paradoxical depolarization of HypoPP fibers in low K+. A major unanswered question is whether the homologous R/X mutations in CaV1.1 associated with HypoPP also produce a gating pore current, thereby providing supportive evidence for a common pathomechanism for HypoPP arising from mutations in NaV1.1 or CaV1.1. The overall goal of this project is to gain a greater understanding for the pathologic basis of HypoPP resulting from CaV1.1 mutations. We have used a gene-targeting approach to generate an R528H knockin mutation of CaV1.1 as a model for HypoPP. The Aims of this project are: (1) to extend the phenotypic characterization of the CaV1.1-R528H mouse for features of hypokalemic periodic paralysis, (2) to test the hypothesis that the CaV1.1-R528H channel conducts an anomalous gating pore current (3) to characterize the integrity of Ca2+- release in CaV1.1-R528H muscle fibers, (4) to explore potential disease-modifying agents in the mouse model of CaV1.1-HypoPP. This work will extend our understanding of the pathogenesis for attacks of weakness in HypoPP and will provide a model system to test the efficacy of therapeutic strategies, both as a means to reduce or ameliorate the burden of disease and to provide confirmatory experimental support for the proposed mechanism of disease. PUBLIC HEALTH RELEVANCE: Familial periodic paralysis is a rare disorder of skeletal muscle in which intermittent failure of muscle excitability causes attacks of severe weakness or paralysis lasting for hours to days. The molecular defect has been known for more than a decade to be mutations of either calcium channels or sodium channels, but the mechanisms by which these channel defects cause susceptibility to attacks of weakness are poorly understood and consequently rational therapeutic strategies to modify disease course are lacking. We have developed the only genetically-engineered mouse model of periodic paralysis based on a calcium channel mutation and will use this unique tool to define further the mechanism underlying attacks of paralysis and test potential interventions to modify disease burden.