MOLECULAR BASIS OF LIGAND NA+ CHANNEL INTERACTIONS

该项目的主要目标是：（1）更好地了解 电压门控Na ~+-Na ~+相互作用的分子基础 通道和局部麻醉剂（LAs）和（2）探索相互作用 LA和Na+渗透途径之间的关系。 在某种程度上， 由选择性过滤器、永久离子结合位点和作为 失活门。 包括的LA中有假定的失活 增强剂，如苯并咪唑和三卡因，推定的开放通道 阻滞剂，如可卡因、布比卡因和季铵（QA） 化合物和假定的双重阻滞剂如丁卡因和普鲁卡因。 在这一建议中，我们计划审查结构基础， 区分这三种不同的LA类型。 两个独立的假设 将测试：首先，只有一个单一的受体存在于 Na+渗透途径的所有三种类型的LA和第二，共同的 失活增强剂苯环上的氨基和双 阻断剂优先稳定Na+的失活状态， 频道 将测量全细胞和单通道电流 为了获得关于动力学的详细动力学信息， Na+通道和LA之间的相互作用。 因为离子-离子排斥 在孔内是离子渗透、演示和 进一步表征LA/QA离子的敲除现象， 将获得通过Na+选择性过滤器的流入阳离子， 提供了关键的证据，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mu 1肌肉Na+通道将进行研究， β 1亚单位存在。 随后，两个不同区域的作用 mu 1 Na+通道，包括内部QA结合位点（可能 孔和S6区域内）和失活相关环 （结构域III和IV之间），对LA结合亲和力将进行检查 在非洲爪蟾卵母细胞注射野生型 和mu 1突变mRNA。 总之，这些研究应该提供一个更清晰的 了解LA-Na+通道的相互作用以及 Na+渗透途径中LA/QA结合位点的变化。

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.