Pain-Associated Neuronal Hyperexcitability and Intracellular Calcium Signaling

描述（由申请人提供）： VA和全球都迫切需要更好的疼痛治疗。该提案的目的是验证改进的测定方法，测量伤害感受器兴奋性，以快速开发更有效的疼痛治疗方法。我们的实验室已经证明，已知引起人类疼痛综合征的电压门控钠通道Nav1.7的功能获得变体对刺激的反应更强烈。我们已经表明，当这些通道变体在啮齿动物DRG神经元中异源表达时，它们引起神经元过度兴奋。此外，我们已经表明，与疼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DRG神经元中表达时也引起过度兴奋。在啮齿动物DRG神经元中表达钠通道变体的方法提供了一种有用的体外模型，用于鉴定引起疼痛综合征或增加载体对疼痛综合征的易感性的变体。我们使用全细胞膜片钳电生理学的研究表明，临床上有效缓解疼痛的药物也会降低该模型中的电兴奋性，使该体外系统适用于测试正在开发用于人类临床测试的新型药理学药物。然而，使用全细胞膜片钳电生理学测量电兴奋性，虽然强大和定量，但低通量，生理性较差（需要破坏细胞完整性的操作），并且不允许相同细胞重复暴露于不同治疗。在这个项目中，我们建议开发一个改进的检测系统，将提供一个独特的平台：a）快速测试的离子通道变异的慢性疼痛患者，以确定其对神经元兴奋性的影响;和B）高通量识别疼痛治疗，这将是最有效的背景下，患者自己的遗传档案。

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.