CONOPEPTIDES THAT TARGET TTX-RESISTANT SODIUM CHANNELS

针对 TTX 抗性钠通道的异肽

• 批准号: 6610792
• 负责人: DOJU YOSHIKAMI
• 金额: $ 9.42万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6610792
• 关键词: Rana; Xenopus; action potentials; afferent nerve; autoradiography; binding sites; chickens; conotoxin; electrophysiology; fluorescence microscopy; fluorescent dye /probe; goldfish; laboratory mouse; neuropharmacology; neurotransmitter antagonist; peptide chemical synthesis; protein isoforms; protein localization; protein structure function; sodium channel; spinal ganglion; tetrodotoxin; voltage gated channel

Action potentials in nerve and muscle fibers are mediated by voltage-gated sodium channels (VGSCs). There are several isoforms of these channels, and most are exquisitely sensitive to tetrodotoxin (TTX), the classic blocker of VGSCs. One such isoform is found exclusively in skeletal muscle, and it is targeted by the founding members in the mu-conopeptide family. Recently, new members of mu-conopeptides (provisionally called mu/nu-conopeptides) have been discovered that irreversibly block TTX-resistant VGSCs in sensory and sympathetic neurons of frog. VGSC-antagonists with this specificity are of medical interest since two TTX-resistant VGSC isoforms cloned from mammals are found largely in nociceptive sensory neurons. Thus, these mu/nu-conopeptides may serve as lead ligands in the development of improved analgesics. There are four specific aims: (1) Examine the activity and selectivity of mu/nu-conopeptides by electrophysiological assays of VGSCs in: (a) sensory and autonomic neurons, (b) motor axons, (c) skeletal muscle, and (d) Xenopus oocytes expressing cloned subtypes of VGSCs. (2) Map the functional distribution of TTX-resistant (mu/nu-conopeptide-sensitive) VGSCs in sensory nerves to examine possible differential expression at sensory endings versus the bulk, or conductive, portion of sensory fibers. This will be done by extracellular recording and focal drug application. (3) Examine the functional tructures of the mu/nu-conopeptides by in-vitro mutagenesis, construction of chimeras of various mu/nuand mu-conopeptides, and covalent labeling with moieties that can also serve as reporter groups for use in the next aim. (4) Map the subcellular distribution of labeled mu/nu-conopeptide binding sites along sensory axons and termini by microscopy. This histological study will complement the electrophysiological experiments in Aim 2.

神经和肌肉纤维的动作电位由电压门控钠通道 (VGSC) 介导。 这些通道有多种同工型，大多数对河豚毒素 (TTX)（VGSC 的经典阻断剂）极其敏感。其中一种异构体仅存在于骨骼肌中，并且是 mu-conopeptide 家族创始成员的目标。最近，发现了 mu-conopeptides 的新成员（暂称为 mu/nu-conopeptides），可以不可逆地阻断青蛙感觉和交感神经元中的 TTX 抗性 VGSC。具有这种特异性的 VGSC 拮抗剂具有医学意义，因为从哺乳动物克隆的两种 TTX 抗性 VGSC 亚型主要存在于伤害性感觉神经元中。因此，这些μ/nu-圆锥肽可以作为改进镇痛药开发中的先导配体。 有四个具体目标：（1）通过 VGSC 的电生理测定来检查 mu/nu-conopeptides 的活性和选择性：（a）感觉和自主神经元，（b）运动轴突，（c）骨骼肌，（d） ) 表达 VGSC 克隆亚型的非洲爪蟾卵母细胞。 (2) 绘制感觉神经中 TTX 抗性（mu/nu-conopeptide 敏感）VGSC 的功能分布，以检查感觉末梢与感觉大部分或传导部分可能的差异表达 纤维。这将通过细胞外记录和局部药物应用来完成。 (3) 通过体外诱变、构建各种 mu/nu 和 mu 圆锥肽的嵌合体以及用也可用作下一个目标的报告基团的部分进行共价标记来检查 mu/nu 圆锥肽的功能结构。 (4)通过显微镜绘制标记的μ/nu-圆锥肽结合位点沿着感觉轴突和末端的亚细胞分布。这项组织学研究将补充目标 2 中的电生理学实验。