Neurotoxin interactions with ligand-gated ion channels

本项目的总体目标是确定神经毒素作用于配体门控离子通道的机制和部位。Picrotoxin （PTX）是一种典型的神经毒素，可抑制GABAA和甘氨酸受体。这些蛋白是结构上相关的受体/CI-通道复合物，是中枢神经系统中主要的抑制性神经递质受体。尽管经过多年的研究，PTX的作用部位和作用机制尚不清楚。我们将检验PTX作用于同型甘氨酸和异型GABAA受体通道深处的假设。由于我们的初步结果表明这些有毒物质在重叠但不同的区域相互作用，我们将定义三种神经毒素的结构决定因素：1)微毒素，2)杀虫剂狄氏剂，3)广泛使用的中枢神经系统惊厥剂戊四唑（PTZ）。我们实验室最近的工作表明，这些神经毒素也阻断5HT3受体，这是一种阳离子选择性配体门控离子通道。这些先前未描述的对阳离子选择性通道的影响可能为这些毒性物质的中心作用提供额外的见解。本提案的具体目的是验证以下假设：1)靠近TM2细胞质方面的通道衬里残基是微毒素的作用部位；2)神经毒素的抑制作用取决于TM2结构域氨基酸的物理特性；(3) PTX、dieldrin和PTZ的功能域重叠但又不同；4)神经毒素通过类似于阴离子选择性通道的结构域阻断阳离子选择性5HT3受体。全面了解神经毒素在昆虫和脊椎动物受体中的作用对于进一步开发新的更安全的杀虫剂至关重要。此外，由于这些试剂被认为在离子通道本身中相互作用，我们的分析将提供有关影响离子通道门控和渗透的通道内衬氨基酸特征的基本信息。这一信息可能与配体门控离子通道超家族的几个成员有关。

The overall goal of this project is to determine the mechanism and site of neurotoxin action on ligand-gated ion channels. Picrotoxin (PTX) is the prototypical neurotoxin, and inhibits both GABAA and glycine receptors. These proteins are structurally related receptor/CI- channel complexes, and are the predominant inhibitory neurotransmitter receptors in the central nervous system. Despite years of study, the site and mechanism of action of PTX is unknown. We will test the hypothesis that PTX acts deep in the channel of the homomeric glycine and the heteromeric GABAA receptor. Because our preliminary results suggest these toxic agents interact at overlapping but distinct domains, we will define the structural determinants for three neurotoxins: 1) picrotoxin, 2) the insecticide dieldrin, and 3) the widely used CNS convulsant pentylenetetrazole (PTZ). Recent work from our lab indicates these neurotoxins also block the 5HT3 receptor, a cation-selective ligand-gated ion channel. These previously undescribed effects on cation-selective channels may provide additional insight into the central actions of these toxic agents. The specific aims of this proposal are to test the following hypotheses: 1) that channel-lining residues near the cytoplasmic aspect of TM2 are the site of action of picrotoxin; 2) that neurotoxin inhibition is dependent on the physical characteristics of the amino acids in this TM2 domain; 3) that the functional domains for PTX, dieldrin and PTZ are overlapping but distinct; and 4) that neurotoxins block the cation-selective 5HT3 receptor through a domain similar to that of the anion- selective channels. A complete understanding of neurotoxin actions in both insects and vertebrate receptors is critical for further development of new and safer insecticides. In addition, because these agents are believed to interact in the ion channel itself, our analysis will provide fundamental information about the characteristics of channel-lining amino acids that influence ion channel gating and permeation. This information may be relevant to several members of the ligand-gated ion channel superfamily.