LOCAL ANESTHETICS--RECEPTOR TOPOLOGY AND DRUG DESIGN

该项目的目标是（1）绘制本地的拓扑结构 麻醉（IA）受体的电压门控Na+通道，（2）以确定 LA受体的一级结构，以及（3）设计更好的Na+ LA受体的通道阻断剂。 到目前为止，LAs对Na+的作用 渠道已经被广泛研究。 LA的潜在机制 然而，作用仍然难以捉摸，主要是因为Na+通道表现出快速的 去极化后的时间和电压依赖性构象变化。 在 在这个提议中，我们计划绘制LA受体的拓扑结构， 平衡条件下的蛙毒素（BTX）激活的肌肉Na+通道 使用简单的平面脂质双层技术。 结构/活动 各种LA及其季铵衍生物的关系，以及 将研究季铵化合物。 亲水和 将详细探索LA受体的疏水结合结构域。 本研究将扩展到宏观电流水平的克隆 存在和不存在BTX的GH 3细胞。 此外，我们计划解决 Na+通道中LA受体的一级结构， 光亲和标记、肽图谱和测序。 假定的LA 受体随后将通过以下的缺失和点突变来描述： Na+通道cDNA。 突变的Na+通道将表达于 非洲爪蟾卵母细胞及其对LA的敏感性将在 电压钳位条件。 同时，我们计划合成各种 两亲性LA衍生物，其被认为将显示高亲和力， 对于LA受体，由于增强的疏水相互作用。 在 返回，这些化合物可用于进一步探测受体 拓扑学和用于光亲和标记。 总之，这些实验 如果Na+通道中的LA受体和 可能为我们提供更好的Na+通道阻滞剂。 如此高的亲和力 阻滞剂可能对慢性AS的管理有潜在的益处， 以及顽固性癌症疼痛。

The goals of this project are (1) to map the topology of the local anesthetic (IA) receptor in voltage-gated Na+ channels, (2) to determine the primary structure of the LA receptor, and (3) to design better Na+ channel blockers for this LA receptor. To date, the action of LAs on Na+ channels has been extensively studied. The mechanism underlying the LA action, however, remains elusive mainly because Na+ channels exhibit rapid time- and voltage-dependent conformational changes upon depolarization. In this proposal, we plan to map the topology of the LA receptor in single batrachotoxin (BTX)-activated muscle Na+ channels at equilibrium conditions using a simple planar lipid bilayer technique. The structure/activity relationship of various LAs and their quaternary derivatives as well as quaternary ammonium compounds will be studied. Both hydrophilic and hydrophobic binding domains of the LA receptor will be explored in detail. This study will then be extended to the macroscopic current level in clonal GH3 cells with and without BTX present. In addition, we plan to resolve the primary structure of the LA receptor in Na+ channels first by photoaffinity labelling, peptide mapping, and sequencing. The putative LA receptor will be subsequently delineated by deletion and point mutation of the Na+ channel cDNA. The mutated Na+ channels will be expressed in Xenopus oocytes and their sensitivity toward LAs will be assayed under voltage clamp conditions. Concurrently, we plan to synthesize a variety of amphipathic LA derivatives which supposedly will display high affinities for the LA receptor due to the enhanced hydrophobic interactions. In return, these compounds may be used for further probing the receptor topology and for photoaffinity labelling. Together, these experiments should give us a clearer view if the LA receptor in Na+ channels and possibly provide us better Na+ channel blockers. Such high affinity blockers may be potentially beneficial for the management of chronic as well as intractable cancer pain.