MOLECULAR BASIS OF LIGAND NA+ CHANNEL INTERACTIONS

这个项目的主要目标是：(1)更好地了解 电压门控钠离子间状态依赖相互作用的分子基础 通道和局麻药(LAS)和(2)的相互作用 LAS和Na+渗透途径之间的关系。这条路径，在一定程度上， 由选择性过滤器、永久离子结合部位和砷组成 失活门。在LAS中包括假定的失活 促进剂，如苯佐卡因和三卡因，假定为明渠 阻滞剂，如可卡因、布比卡因和季铵盐(QA) 化合物，以及可能的双重阻滞剂，如丁卡因和普鲁卡因。 在这项建议中，我们计划研究以下结构基础 区分了这三种不同的LA类型。两个不同的假设 将被测试：首先，只有一个受体存在于 三种类型LAS和第二种常见LAS的Na+渗透途径 灭活促进剂苯环上的氨基及其对映体 阻滞剂优先稳定Na+的失活状态 频道。将测量全电池和单通道电流 为了获得关于动力学的详细动力学信息 Na+通道与LAS的相互作用。因为离子-离子排斥力 在孔洞内是离子渗透、演示和 LA/QA离子敲除现象的进一步表征 通过Na+选择性过滤器流入的阳离子将被获得 提供关键证据，证明LA结合位点确实位于 在Na+渗透途径中。同时，我们将描绘洛杉矶- 分子水平上的通道相互作用。首先，LA绑定 对克隆的MU1肌Na+通道的研究 存在Beta1亚基。随后，两个独立区域的作用 包括内部QA结合位点(可能是 在孔和S6区域内)和失活相关环 (在结构域III和IV之间)，将检查LA上的结合亲和力 非洲爪哇卵母细胞注射野生型大片技术的研究 和MU1突变型mRNAs。总而言之，这些研究应该提供一个更清晰的 对LA-Na+通道相互作用及其去向的理解 Na+渗透途径中LA/QA结合部位的变化。

The broad objectives of this project are: (1) to understand better the molecular basis of state-dependent interactions between voltage-gated Na channels and local anesthetics (LAs) and (2) to explore the interplay between LAs and the Na+ permeation pathway. This pathway, in part, consists of a selectivity filter, permenant ion binding sites and as inactivation gate. Among LAs included are putative inactivation enhancers, such as benzocaine and tricaine, putative open-channel blockers, such as cocaine, bupivacaine, and quaternary ammonium (QA) compounds, and putative dual blockers such as tetracaine and procaine. In this proposal, we plan to examine the structural basis that distinguishes these three distinct LA types. Two separate hypotheses will be tested: first, only one single receptor is present within the Na+ permeation pathway for all three types of LAs and second, the common amino group on the phenyl ring of inactivation enhancers and dual blockers preferentially stabilizes the inactivated state of the Na+ channel. Both whole-cell and single channel currents will be measured in order to obtain detailed kinetic information on the dynamic interactions between Na+ channels and LAs. Because ion-ion repulsion within the pore is a common trait for ion permeation, demonstration and further characterization of a knock-out phenomenon of LA/QA ions by the inflowing cations through the Na+ selectivity filter will be obtained to provide crucial evidence that the LA binding site is indeed located within the Na+ permeation pathway. Concurrently, we will delineate LA- channel interactions at the molecular level. At first, the LA binding toward cloned mu1 muscle Na+ channels will be studied with and without Beta1 subunit present. Subsequently, the roles of two separate regions of mu1 Na+ channels, including the internal QA binding site (probably within the pore and S6 regions) and the inactivation-related loop (between domain III and IV), on LA binding affinities will be examined by the macropatch technique in Xenopus oocytes injected with wild-type and mu1 mutant mRNAs. Together, these studies should provide a clearer understanding of LA-Na+ channel interactions as well as the whereabouts of the LA/QA binding site within the Na+ permeation pathway.