LOCAL ANESTHETICS--RECEPTOR TOPOLOGY AND DRUG DESIGN

该项目的目标是(1)绘制本地网络的拓扑图 电压门控性钠通道的麻醉剂(IA)受体，(2)测定 LA受体的一级结构，以及(3)设计更好的Na+ 这种LA受体的通道阻滞剂。到目前为止，LAS对Na+的作用 经络已被广泛研究。LA背后的机制 然而，作用仍然难以捉摸，主要是因为钠离子通道表现出快速的 去极化时依赖于时间和电压的构象变化。在……里面 在这项提议中，我们计划将LA受体的拓扑图 平衡状态下巴曲霉毒素(BTX)激活的肌肉Na+通道 使用一种简单的平面脂质双层技术。结构/活动 各种LAS与其四元衍生物的关系以及 将对季铵化合物进行研究。无论是亲水性的还是 我们将详细探讨LA受体的疏水结合域。 这项研究随后将扩展到克隆的宏观电流水平 有无BTX存在的GH3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。推定的LA 受体随后将通过缺失和点突变来描绘 Na+通道基因。突变的Na+通道将在 非洲爪哇卵母细胞及其对LAS的敏感性将在 电压钳位条件。同时，我们计划合成各种不同的 两亲性LA衍生物，据推测将显示出高亲和力 对于LA受体，由于增强的疏水相互作用。在……里面 返回，这些化合物可用于进一步探测受体 用于拓扑和光亲和标记。总而言之，这些实验 应该能让我们更清楚地看到Na+通道和 可能会给我们提供更好的钠通道阻滞剂。如此高的亲和力 阻滞剂可能对慢性AS的治疗有潜在的益处 以及顽固的癌症疼痛。

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.