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

描述（由申请人提供）：低钾性周期性麻痹（HypoPP）是一种显性遗传性骨骼肌疾病，其中纤维电兴奋性的间歇性失效导致虚弱的反复发作。癫痫与下列疾病有关： 低钾血症（K+ < 3 mM），可能由碳水化合物摄入、运动或压力引发。HypoPP的分子缺陷是异质性的，其中60%的家族在编码L型Ca通道CaV1.1的CACNA 1 S中具有错义突变，另外20%在编码电压门控Na通道NaV1.4的SCN 4A中具有错义突变，其余的未确定。尽管这一科学进步，致病的基础上，短暂的攻击纤维去偏振与兴奋性的损失是没有完全建立。奇怪的是，NaV1.4中的所有8个突变和CaV1.1中的7个突变中的6个都发生在S4电压传感器结构域的精氨酸残基上。到目前为止，在切开的卵母细胞中研究的所有6种NaV1.4-HypoPP突变都揭示了在超极化电位下激活的小的异常阳离子电流，其通过突变的S4片段和通道蛋白之间的“门控孔”进行传导。我们最近报道了NaV1.4-R669 H小鼠肌纤维中的门控孔电流。这种门控孔电导被假设为内向电流的来源，该内向电流触发了低K+下HypoPP纤维的反常去极化。一个主要的未回答的问题是是否同源R/X突变CaV1.1与HypoPP也产生门控孔电流，从而提供了一个共同的病理机制HypoPP产生的NaV1.1或CaV1.1突变的支持性证据。该项目的总体目标是获得对CaV1.1突变导致的HypoPP的病理基础的更好理解。我们已经使用基因靶向方法来产生CaV1.1的R528 H敲入突变作为HypoPP的模型。该项目的目标是：（1）扩展CaV1.1-R528 H小鼠的低钾性周期性麻痹特征的表型表征，（2）检验CaV1.1-R528 H通道传导异常门控孔电流的假设，（3）表征CaV1.1-R528 H肌纤维中Ca 2+释放的完整性，（4）在CaV1.1-HypoPP小鼠模型中探索潜在的疾病缓解剂。这项工作将扩展我们对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疗效，既作为减少或改善疾病负担的手段，又为拟议的疾病机制提供验证性实验支持。 公共卫生相关性：家族性周期性麻痹是一种罕见的骨骼肌疾病，其中肌肉兴奋性的间歇性衰竭导致持续数小时至数天的严重虚弱或瘫痪。十多年来，分子缺陷被认为是钙通道或钠通道的突变，但这些通道缺陷导致对虚弱发作易感性的机制知之甚少，因此缺乏合理的治疗策略来改变疾病进程。我们已经开发出了唯一的基于钙通道突变的周期性麻痹基因工程小鼠模型，并将使用这种独特的工具来进一步定义瘫痪发作的机制，并测试潜在的干预措施来改变疾病负担。

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.