Local anesthetic receptor in peripheral Na+ channels

描述（由申请方提供）：局部麻醉剂（LA）阻断电压门控Na+通道，并在局部麻醉期间可逆地阻碍运动和感觉功能。因此，外周Na+通道中的LAs受体是治疗的重要靶标。我们已经证明，对慢性疼痛有效的药物，如抗抑郁药阿米替林和度洛沙汀、抗心律失常药氟卡尼、抗心绞痛药雷诺嗪和抗惊厥药美西律，都优先通过LA受体阻断持续性晚期Na+电流。这种晚期Na+电流流经失活缺陷型Na+通道的开放状态，已知会引起慢性疼痛，如在遗传性Nav1.7通道病中发现的。进一步的证据表明，在治疗血浆浓度下，度洛沙汀和雷诺嗪在阻滞晚期Nav1.7 Na+电流方面比骨骼肌Nav1.4或心脏Nav1.5对应物更有效。因此，我们假设这些治疗药物靶向失活缺陷型Nav1.7和/或Nav1.8 Na+通道的开放状态，以获得缓解疼痛的疗效。我们的长期目标是在持续开放的Nav1.7和Nav1.8 Na+通道中确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剂用作野生型和失活缺陷型Nav1.7和Nav1.8 Na+通道中受体的分子探针。五个具体目标将指导这项工作：1。测量这些疼痛治疗剂对Nav1.7和Nav1.8 Na+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 Na+电流，3，使用培养的DRG神经元验证Nav1.7和Nav1.8 Na+通道的有效阻断的生理相关性，4，确定Nav1.7和Nav1.8 Na+通道中独特的药物结合残基，以及5，使用计算机建模来重建Nav1.7和Nav1.8开放Na+通道内腔中的该药物结合位点。具体来说，我们计划通过瞬时转染在哺乳动物Hek 293 t细胞中表达Nav1.7和Nav1.8 Na+通道。将在野生型和失活缺陷型Nav1.7和Nav1.8突变型Na+通道中测定静息、开放和失活阻断的50%抑制药物浓度（IC 50）。将使用含有天然细胞内成分的培养大鼠DRG神经元，结合将野生型或突变型Nav1.7和Nav1.8 Na+通道转染至这些细胞中，进行药物效价验证。失活缺陷型Nav1.7和Nav1.8通道中药物结合残基的置换将通过定点诱变进行，突变体将进行药物筛选。将通过计算机建模可视化内腔内药物结合位点的差异。总之，我们的研究将揭示这些不同的药物如何在其治疗相关浓度下通过共享的度洛沙汀/雷诺嗪/LA受体选择性靶向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治疗的发展提供新的策略。

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.