Pain-Associated Neuronal Hyperexcitability and Intracellular Calcium Signaling

描述(由申请人提供)： 退伍军人事务部和全球都迫切需要更好的疼痛治疗方法。这项建议的目的是验证改进的测试方法，这些方法可以测量伤害性感受器的兴奋性，从而迅速开发出更有效的疼痛治疗方法。我们的实验室已经证明，已知会导致人类疼痛综合征的电压门控钠通道Nav1.7的功能增益变体对刺激反应更强烈。我们已经证明，当这些通道变体在啮齿动物背根神经节神经元中异源表达时，它们会引起神经元的超兴奋性。此外，我们还发现，与痛性周围神经病变相关的电压门控钠通道Nav1.8和Nav1.9的变体在啮齿动物背根神经节神经元中表达时也会引起高度兴奋。在啮齿动物背根神经节神经元中表达钠通道变体的方法为识别导致或增加携带者对发展中的疼痛综合征的易感性的变体提供了一个有用的体外模型。我们使用全细胞膜片钳电生理学的研究表明，在这个模型中，临床上有效的止痛药物也可以降低电兴奋性，使这个体外系统适合于测试正在开发的用于临床测试的新型药理药物。然而，使用全细胞膜片钳电生理学测量电兴奋性，虽然功能强大且定量，但吞吐量低，生理学较差(需要破坏细胞完整性的操作)，并且不允许同一细胞重复暴露于不同的处理。在这个项目中，我们建议开发一种改进的测试系统，该系统将提供一个独特的平台，用于：a)快速测试慢性疼痛患者的离子通道变体，以确定它们对神经元兴奋性的影响；以及b)根据患者自身的遗传特征高通量识别最有效的疼痛疗法。

DESCRIPTION (provided by applicant): Better treatments for pain are urgently needed both within the VA and globally. The objective of this proposal is to validate improved assays that measure nociceptor excitability toward the rapid development of more effective treatments for pain. Our laboratory has demonstrated that gain-of-function variants of the voltage- gated sodium channel Nav1.7 known to cause human pain syndromes respond more strongly to stimuli. We have shown that when these channel variants were heterologously expressed in rodent DRG neurons they elicit neuronal hyperexcitability. In addition, we have shown that variants of voltage-gated sodium channels Nav1.8 and Nav1.9 associated with painful peripheral neuropathies also elicit hyperexcitability when expressed in rodent DRG neurons. The approach of expressing sodium channel variants in rodent DRG neurons provides a useful in vitro model toward identifying variants that cause, or increase the susceptibility of carriers to developing, pain syndromes. Our studies using whole-cell patch-clamp electrophysiology have shown that drugs that are clinically effective in mitigating pain also reduce electrical excitability in this model, making this in vitro system suitable for testing novel pharmacological agents that are being developed for clinical testing in humans. However, measurement of electrical excitability using whole-cell patch-clamp electrophysiology, although powerful and quantitative, is low-throughput, less physiological (requiring manipulations that disrupt cell integrity), and does not permit repeated exposures of the same cell to different treatments. In this project we propose to develop an improved assay system that would provide a unique platform for: a) rapid testing of ion channel variants from patients with chronic pain to determine their effect on neuronal excitability; and b) high-throughput identification of pain therapeutics that would be most effective in the context of the patient's own genetic profile.