Local anesthetic receptor in peripheral Na+ channels

描述(申请人提供)：局部麻醉药(LAS)在局部麻醉过程中阻断电压门控的Na+通道，并可逆性地阻碍运动和感觉功能。因此，外周Na+通道上的LAS受体是治疗的重要靶点。我们发现，对慢性疼痛有效的药物，如抗抑郁药阿米替林和度洛西汀、抗心律失常氟卡胺、抗心绞痛雷诺嗪和抗惊厥药美西律，都优先通过LA受体阻断持续的晚期Na+电流。这种晚期的Na+电流流经失活缺陷的Na+通道的开放状态，已知会导致慢性疼痛，如遗传性Nav1.7通道病。进一步的证据表明，在治疗血浆浓度下，度洛西汀和雷诺嗪在阻断晚期Nav1.7 Na+电流方面比骨骼肌Nav1.4或心脏NaV1.5电流更有效。因此，我们假设这些疗法针对的是失活缺陷的Nav1.7和/或Nav1.8钠离子通道的开放状态，以达到缓解疼痛的效果。我们的长期目标是在持续开放的Nav1.7和Nav1.8 Na+通道中界定这些药物的独特受体。为了实现这一目标，我们计划将上述疼痛治疗药物作为分子探针应用于野生型和失活缺陷的Nav1.7和Nav1.8 Na+通道。这项工作将以五个具体目标为指导：1.用这些止痛药物测量Nav1.7和Nav1.8钠通道的静息和失活阻断，2，确定这些药物是否选择性地阻断持续的晚期Nav1.7和/或Nav1.8钠电流，3，利用培养的DRG神经元验证Nav1.7和Nav1.8钠通道的有效阻断的生理学相关性，4，识别Nav1.7和Nav1.8钠通道中独特的药物结合残基，以及5，使用计算机模拟在Nav1.7和Nav1.8开放的Na+通道的内腔内重建该药物结合部位。具体地说，我们计划通过瞬时转基因在哺乳动物Hek293t细胞中表达Nav1.7和Nav1.8 Na+通道。静息、开放和失活阻断的50%抑制药物浓度(IC50)将在野生型和失活缺陷的Nav1.7和Nav1.8突变的Na+通道中确定。药物效力的验证将使用含有天然细胞内成分的培养的大鼠背根神经节神经元，并将野生型或突变的Nav1.7和Nav1.8 Na+通道导入这些细胞。灭活缺陷的Nav1.7和Nav1.8通道中的药物结合残基的替换将通过定点突变进行，突变体将接受药物筛选。内腔内药物结合部位的差异将通过计算机建模可视化。总之，我们的研究将揭示这些不同的药物如何通过共享的度洛西汀/雷诺嗪/LA受体在其治疗相关浓度下选择性地靶向Nav1.7和/或Nav1.8 Na+通道的开放状态。这些信息将为疼痛选择性疗法的发展提供新的战略。

DESCRIPTION (provided by applicant): Local anesthetics (LAs) block voltage-gated Na+ channels and impede both motor and sensory functions reversibly during local anesthesia. The receptor for LAs in peripheral Na+ channels is therefore an important target for therapeutics. We have shown that drugs effective for chronic pain, such as antidepressants amitriptyline and duloxetine, antiarrhymthic flecainide, antianginal ranolazine, and anticonvulsant mexiletine, all preferentially block persistent late Na+ currents via the LA receptor. Such late Na+ currents that flow through the open state of inactivation-deficient Na+ channels are known to cause chronic pain, as found in inherited Nav1.7 channelopathies. Further evidence indicates that duloxetine and ranolazine at their therapeutic plasma concentrations are more potent in the block of late Nav1.7 Na+ currents than those of skeletal muscle Nav1.4 or cardiac Nav1.5 counterparts. We therefore hypothesize that these therapeutics target the open state of inactivation-deficient Nav1.7 and/or Nav1.8 Na+ channels for their efficacy in pain relief. Our long-term objective is to delimit the unique receptor for these drugs in persistently open Nav1.7 and Nav1.8 Na+ channels. Toward this goal, we plan to apply above pain therapeutics as molecular probes for their receptors in wild-type and inactivation-deficient Nav1.7 and Nav1.8 Na+ channels. Five specific aims will guide this work: 1. measure the resting and inactivated block of Nav1.7 and Nav1.8 Na+ channels by these pain therapeutics, 2, determine if these drugs block persistent late Nav1.7 and/or Nav1.8 Na+ currents selectively, 3, validate the physiological relevance of the potent block of Nav1.7 and Nav1.8 Na+ channels using cultured DRG neurons, 4, identify the unique drug-binding residues in Nav1.7 and Nav1.8 Na+ channels, and 5, use computer modeling to reconstruct this drug binding site within the inner cavity of Nav1.7 and Nav1.8 open Na+ channels. Specifically, we plan to express Nav1.7 and Nav1.8 Na+ channels in mammalian Hek293t cells by transient transfection. The 50% inhibitory drug concentration (IC50) of resting-, open-, and inactivated block will be determined in wild-type and in inactivation-deficient Nav1.7 and Nav1.8 mutant Na+ channels. Validation of drug potency will be conducted using cultured rat DRG neurons with native intracellular ingredients in conjunction with transfection of wild-type or mutant Nav1.7 and Nav1.8 Na+ channels into these cells. Substitutions of drug-binding residues in inactivation-deficient Nav1.7 and Nav1.8 channels will be performed by site-directed mutagenesis and mutants will be subjected to drug screening. Differences in the drug binding site within the inner cavity will be visualized by computer modeling. Together, our studies will reveal how these diverse drugs selectively target the open state of Nav1.7 and/or Nav1.8 Na+ channels via the shared duloxetine/ranolazine/LA receptor at their therapeutic relevant concentrations. Such information will provide new strategies for the development of pain-selective therapeutics.