Pathophysiology of Myotonia and Periodic Paralysis

项目摘要/摘要 周期性瘫痪和肌强直是骨骼肌的离子通道病，伴有严重的 持续数小时至数天的虚弱和依赖活动的肌肉僵硬。这个项目的长期目标是 促进我们对这些肌肉兴奋性障碍的发病机制的认识，并应用于 治疗干预措施的设计和临床前测试方面的知识。 在建立脑血管疾病生物物理缺陷之间的因果关系方面已经取得了很大进展 突变通道与临床表型的关系。例如，超过80个错义突变已经在 NaV1.4钠通道，我们已经通过功能表达研究和模拟 纤维兴奋性，随着功能变化(如失活受损)而发生的突变导致的高血钾 周期性麻痹(HyperPP)伴肌强直。另一种选择是，低钾周期性的NaV1.4突变 麻痹(HypoPP)是电压传感器结构域的S4段中的所有R/X替换，它们共享一个共同的 功能缺陷--异常的门孔泄漏电流。在所有形式的周期性瘫痪中，暂时性 虚弱发作的原因是𝑉𝑟𝑒௦௧持续的去极化和兴奋性的丧失，这通常是触发的 受压力、饮食(碳水化合物、盐含量、禁食)、寒冷温度或锻炼的影响。它们的作用机制 在突变通道的静态缺陷的设置中，这些触发器使𝑉𝑟𝑒௦௧不稳定，是基本开放的 这不仅是该领域存在的问题，也是进行治疗干预的机会。一个主要的障碍 进展一直是受影响肌肉的稀缺性。我们创造了三个基因敲入突变小鼠模型 具有强大的HyperPP(NaV1.4-M1592V)或HypoPP(NaV1.4-R669H；CaV1.1-R528H)表型的PP。 这些小鼠模型导致了对疾病机制的新见解(例如，从酸中毒中恢复是一种有效的 并导致了新的治疗干预措施，目前正在进行临床试验(布美他尼 抑制NKCC1共转运体可防止HypoPP)。 我们将通过重点研究离子梯度的影响来扩展我们对周期性瘫痪的研究。 细胞外[K]o的变化是低PP(低)或高PP(高)的既定触发因素，但相对较小的是 已知PP中的Na和Cl-移动。有限的人体数据表明，在一段时间内，[Na]in急剧上升 HypoPP为HyperPP或慢性高[Na]In。此外，我们还表明，完全减少氯离子的内流 防止HypoPP攻击。我们已经开发出改进的离子选择微电极，与 我们的基因敲入突变小鼠的独特资源将使我们能够(1)表征肌肉纤维Na和Cl- 静息和PP攻击期间的内容，(2)定义特定离子传输系统(突变体)的贡献 NaV1.4，NKCC1，Na/K-ATPase，Cl-Exchangers)在肌肉通道病中设定离子浓度，(3) 定义PP中离子梯度扰动的功能后果，基于计算模型和 模拟，以及(4)将这些见解用于疾病修正干预措施的设计和临床前测试。 。

Project Summary / Abstract Periodic paralysis and myotonia are ion channelopathies of skeletal muscle with debilitating episodes of severe weakness lasting hours to days and activity-dependent muscle stiffness. The long-term goal of this project is to advance our understanding of disease mechanism in these disorders of muscle excitability and to apply this knowledge in the design and pre-clinical testing of therapeutic interventions. Much progress has been made in establishing a causal relationship between the biophysical defect of a mutant channel and the clinical phenotype. For example, over 80 missense mutations have been identified in the NaV1.4 sodium channel, and we have shown by functional expression studies, coupled with simulations of fiber excitability, that mutations with gain of function changes (e.g. impaired inactivation) cause hyperkalemic periodic paralysis (HyperPP) with myotonia. Alternatively, the NaV1.4 mutations in hypokalemic periodic paralysis (HypoPP) are all R/X substitutions in S4 segments of voltage sensor domains that share a common functional defect - the anomalous gating pore leakage current. In all forms of periodic paralysis, the transient attacks of weakness result from sustained depolarization of 𝑉𝑟𝑒௦௧ and loss of excitability, which are often triggered by stress, diet (carbohydrate, salt content, fasting), cold temperature, or exercise. The mechanisms by which these triggers destabilize 𝑉𝑟𝑒௦௧, in the setting of a static defect for a mutant channel, are fundamental open questions in the field and also represent opportunities for therapeutic intervention. A major impediment to progress has been the scarce availability of affected muscle. We created three knock-in mutant mouse models of PP that have robust phenotypes for HyperPP (NaV1.4-M1592V) or HypoPP (NaV1.4-R669H; CaV1.1-R528H). These mouse models have led to new insights on disease mechanism (e.g. recovery from acidosis is a potent trigger of HypoPP) and have led to novel therapeutic interventions that are now in clinical trials (bumetanide inhibition of the NKCC1 cotransporter prevents HypoPP). We will extend our investigations of periodic paralysis by focusing on the impact of ion gradients. Changes in extracellular [K+]o are established triggers for HypoPP (low) or HyperPP (high), but relatively little is known about Na+ and Cl- shifts in PP. Limited human data suggest an acute rise of [Na+]in during an episode of HyperPP or chronically high [Na+]in for HypoPP. In addition, we showed that reducing Cl- influx completely prevents HypoPP attacks. We have developed improved ion-selective microelectrodes, that in combination with the unique resource of our knock-in mutant mice, will enable us to (1) characterize muscle fiber Na+ and Cl- content at rest and during an attack of PP, (2) define the contribution of specific ion transport systems (mutant NaV1.4, NKCC1, Na/K-ATPase, Cl- exchangers) in setting ion concentrations in muscle channelopathies, (3) define the functional consequences of ion gradient perturbations in PP, based on computational modeling and simulation, and (4) use these insights in the design and pre-clinical testing of disease-modifying interventions. .