Local anesthetic receptor in peripheral Na+ channels

外周Na通道中的局部麻醉受体

• 批准号: 8107925
• 负责人: GING K WANG
• 金额: $ 33.22万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8107925
• 关键词: Affinity; Amitriptyline; Analgesics; Anesthetics; Anticonvulsants; Antidepressive Agents; Binding; Cardiac; Cells; Clinic; Clinical; Computer Simulation; Data; Development; Drug Binding Site; Drug Receptors; Drug effect disorder; Drug usage; Engineering; Flecainide; Future; Goals; Inherited; Inhibitory Concentration 50; Lidocaine; Local Anesthetics; Local anesthesia; Measures; Mexiletine; Molecular Conformation; Molecular Probes; Motor; Muscle; Neurons; Nociception; Pain; Pain management; Patch-Clamp Techniques; Peripheral; Pharmaceutical Preparations; Physiological; Plasma; Play; Preclinical Drug Evaluation; Protein Isoforms; Rattus; Resistance; Rest; Role; Sensory; Site; Site-Directed Mutagenesis; Skeletal Muscle; Sodium Channel; Structural Models; Therapeutic; Therapeutic Index; Therapeutic Uses; Transfection; Validation; Work; base; chronic pain; design; duloxetine; in vivo; mutant; novel; painful neuropathy; ranolazine; receptor; research study; therapeutic target; voltage

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. PUBLIC HEALTH RELEVANCE: The local anesthetic receptor in peripheral sodium channels is an important target for clinical drugs used in local anesthesia and in pain management. Recent evidence suggests that pain therapeutics such as duloxetine may act as analgesics to silence persistent late Na+ currents via a duloxetine/local anesthetic shared receptor. Our goals are to delimit such a receptor in peripheral Nav1.7 and Nav1.8 channels, to resolve the drug/receptor interactions during state transitions, and to explore this receptor site for novel pain therapeutics.

描述（由申请人提供）：局部麻醉剂（LAs）在局部麻醉期间阻断电压门控Na+通道，可逆地阻碍运动和感觉功能。因此，外周Na+通道中LAs的受体是治疗的重要靶点。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西汀，抗心律失常的氟化胺，抗心绞痛的诺拉嗪和抗惊厥的美西汀，都优先阻断通过LA受体的持续的晚期Na+电流。这种晚期Na+电流流经失活缺陷Na+通道的开放状态，已知会引起慢性疼痛，正如在遗传性Nav1.7通道病变中发现的那样。进一步的证据表明，治疗血浆浓度的度洛西汀和雷诺嗪比骨骼肌Nav1.4或心脏Nav1.5更有效地阻断晚期Nav1.7 Na+电流。因此，我们假设这些疗法针对失活缺陷的Nav1.7和/或Nav1.8 Na+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疗法应用于野生型和失活缺陷的Nav1.7和Nav1.8 Na+通道中受体的分子探针。指导这项工作的具体目标有五个：2，确定这些药物是否有选择性地阻断持续的晚期Nav1.7和/或Nav1.8 Na+电流，3，利用培养的DRG神经元验证Nav1.7和Nav1.8 Na+通道有效阻断的生理相关性，4，鉴定Nav1.7和Nav1.8 Na+通道中独特的药物结合残基，5，利用计算机建模重建Nav1.7和Nav1.8内腔内开放Na+通道内的药物结合位点。具体来说，我们计划通过瞬时转染在哺乳动物Hek293t细胞中表达Nav1.7和Nav1.8 Na+通道。将在野生型和失活缺陷的Nav1.7和Nav1.8突变Na+通道中测定静息、开放和失活阻断的50%抑制药物浓度（IC50）。药物效力的验证将使用含有天然细胞内成分的培养大鼠DRG神经元，并将野生型或突变型Nav1.7和Nav1.8 Na+通道转染到这些细胞中。失活缺陷的Nav1.7和Nav1.8通道中的药物结合残基将通过定点诱变进行替换，突变体将经过药物筛选。内腔内药物结合位点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在治疗相关浓度下，通过共享的度洛西汀/雷诺嗪/LA受体，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治疗的发展提供新的策略。