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

低钾性周期性麻痹（HypoPP）是一种显性遗传性骨骼肌疾病，其中由于纤维兴奋性的间歇性失效而导致虚弱的反复发作。低钾血症（K+ <3 mM）会导致Epilepsy，通常由碳水化合物摄入、运动或压力引发。HypoPP的分子缺陷是异质性的，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，20%的家族在编码电压门控Na通道NaV1.4的SCN 4A中具有错义突变。尽管有这一科学进步，但纤维去极化和兴奋性丧失的短暂发作的致病基础尚未完全建立，缺乏有效的临床干预措施。拟议的研究旨在促进我们对CaV1.1 HypoPP突变引起的功能缺陷的理解，并将这些信息应用于模型模拟中，以获得对疾病机制的见解，从而确定实验可测试的疾病修饰策略。NaV1.4中的所有10个HypoPP突变和CaV1.1中的11个HypoPP突变中的9个发生在电压传感器结构域（VSD）的S4段的精氨酸残基处。对于NaV1.4 HypoPP，这些R/X突变使通道泄漏，因为小的异常“门控孔电流”。通过同源性，已经提出了CaV1.1 HypoPP的类似缺陷，但实验证实有限。在本项目的前一个周期中，我们在我们的R528 H敲入突变型HypoPP小鼠模型中检测到CaV1.1门控孔电流。这一发现支持了这样的观点，即异常门控孔电流是NaV1.4和CaV1.1 HypoPP突变通道之间的共同特征，从而解释了任一通道的突变如何产生相同的临床表型。CaV1.1-R528 H小鼠具有稳健的HypoPP表型，并且是研究环境触发物引起兴奋性丧失和虚弱的机制的独特资源。例如，我们最近发现， 从酸中毒中恢复是R528 H小鼠比目鱼肌力量暂时丧失的有效触发因素，这可能 提供了第一个关于为什么在HypoPP患者剧烈运动后经常发生虚弱发作的见解。的 完全分化的HypoPP肌纤维的可用性为研究兴奋-收缩提供了极好的系统 偶联和Ca ~（2+）释放在纯合R528 H纤维中被显著抑制。在其他的初步研究中，我们已经实现了50倍的CaV1.1在非洲爪蟾卵母细胞中的表达增加共表达Stac 3，伴侣，促进CaV贩运到膜。这种新的高表达系统现在可以确定其他CaV1.1 HypoPP突变，特别是S4中R残基上的两个非典型突变是否也支持门控孔电流。CaV1.1-R528 H HypoPP小鼠和非洲爪蟾卵母细胞中CaV1.1的膜表达增强将用于解决以下特定目的：（1）检验异常门控孔电流是与HypoPP相关的CaV1.1突变共同的病理机制的假设，（2）在HypoPP运动后虚弱的pH变化模型中，检验以下假设：Cl-梯度的变化导致对去极化诱导的兴奋性丧失的敏感性，限制Cl-积聚的策略降低了虚弱的严重程度。(3)测试受损的Ca 2+释放是否是CaV1.1 R528 H肌肉中的普遍缺陷，并探索该缺陷是否是通道突变所固有的。

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 excitability. Episodes occur in association with hypokalemia (K+ <3 mM) and are often 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, and 20% have missense mutations in SCN4A encoding the voltage-gated Na channel NaV1.4. Despite this scientific advance, the pathogenic basis for the transient attacks of fiber depolarization and loss of excitability is not fully established, and effective clinical interventions are lacking. The proposed studies are designed to advance our understanding of the functional defects caused by CaV1.1 HypoPP mutations and to apply this information in model simulations to gain insights on disease mechanism and thereby identify experimentally testable strategies for disease modification. All 10 HypoPP mutations in NaV1.4 and 9 of 11 in CaV1.1 occur at arginine residues in S4 segments of voltage-sensor domains (VSD). For NaV1.4 HypoPP, these R/X mutations make channels leaky because of a small anomalous “gating pore current”. By homology, a similar defect has been proposed for CaV1.1 HypoPP, but experimental confirmation has been limited. In the prior cycle of this project, we detected a CaV1.1 gating pore current in our R528H knockin mutant mouse model of HypoPP. This finding supports the notion that the anomalous gating pore current is a feature in common between NaV1.4 and CaV1.1 HypoPP mutant channels, and thereby explains how mutations of either channel can produce the same clinical phenotype. The CaV1.1-R528H mice have a robust HypoPP phenotype and are a unique resource for investigating mechanisms by which environmental triggers elicit loss of excitability and weakness. For example, we recently discovered that recovery from acidosis is a potent trigger for a transient loss of force in soleus muscle of R528H mice, which may provide the first insight on why attacks of weakness frequently occur after strenuous exercise in HypoPP patients. The availability of fully-differentiated HypoPP muscle fibers provides an excellent system to study excitation-contraction coupling, and Ca2+ release is markedly suppressed in homozygous R528H fibers. In other preliminary studies, we have achieved a 50-fold increase of CaV1.1 expression in Xenopus oocytes by co-expressing Stac3, a chaperone that promotes CaV trafficking to the membrane. This new high-expression system now makes it possible to determine whether the other CaV1.1 HypoPP mutations, especially the two atypical mutations not at R residues in S4, also support a gating pore current. The CaV1.1-R528H HypoPP mouse and the enhanced membrane expression of CaV1.1 in Xenopus oocytes will be used to address the following Specific Aims: (1) Test the hypothesis that an anomalous gating pore current is a pathomechanism in common with CaV1.1 mutations associated with HypoPP, (2) In the pH-shift model for post-exercise weakness in HypoPP, test the hypothesis that a shift in the Cl- gradient causes susceptibility to depolarization-induced loss of excitability and that maneuvers to limit Cl- accumulation reduce the severity of weakness. (3) Test whether impaired Ca2+ release is a pervasive defect in CaV1.1 R528H muscle and explore whether the defect is intrinsic to the channel mutation.