STRUCTURAL ELEMENTS OF NICOTINIC ACETYLCHOLINE RECEPTORS

最近，我们对生物学的认识有了新的进展。 烟碱型乙酰胆碱受体可能在 大脑，克隆这些受体的基因给了我们 研究这些受体的详细分子机制的工具[l]。 已知尼古丁受体参与成瘾过程[2]， 并被认为是精神分裂症的影响[3，4]。他们有 也被证明对认知过程和记忆很重要。 烟碱激动剂正被开发作为治疗 老年痴呆症[5]。沿着人们对 烟碱受体在脑功能中可能发挥的功能作用， 来欣赏存在于细胞中的多种受体亚型， 个脑袋这一建议将扩大我们的理解，如何一个关键的 生理特性，二价离子的渗透性，受到调节 在受体亚型（即亚基组合）的水平上， 在特定的蛋白质结构域方面。二价离子渗透性可以是 与学习和记忆相关的神经元可塑性所需的 并且还可能产生兴奋性毒性。 我们将在非洲爪蟾卵母细胞中表达克隆的烟碱受体， 嵌合和突变亚基的研究，我们将确定分子 调节钙渗透性的元素，以及分子 与受体的使用依赖性抑制有关的元素。 通过评估调节这些因素之间的关系， 二价离子渗透性，以及与敏感性相关的那些 对于特定的拮抗剂，可以定义 可以靶向特定的功能重要的受体亚型， 避开那些对认知过程很重要的受体， 那些参与成瘾过程，或选择性地阻止那些 可能会使细胞处于毒性的危险中。 将进行受体生理学和药理学的分析 无论是在全细胞电流的水平上， 单通道属性。对相关系列的研究 双功能抑制剂将允许抑制性结合的处置 待评价的受体复合物内的位点。 我们广泛的初步数据为我们提供了强有力的基础， 实验设计的方法和候选序列， 重要的结构域。我们有证据表明二价离子 渗透性由肌肉受体的γ亚单位调节， 神经元受体的α 5亚单位。该模型将直接 用反义基因敲除实验进行评价， 这些亚基在天然受体中的功能影响。 该提案中的实验将提供重要的新见解， 大脑的烟碱受体在生物物理学上 性质，它们作为治疗靶点的潜力，以及它们之间的关系 特定的功能特性和药物敏感性之间的关系。

Recently new advances have been made in our understanding of the functional roles which nicotinic acetylcholine receptors may play in the brain, and the cloning of the genes for these receptors has given us the tools to study the detailed molecular mechanism of these receptors [l]. Nicotinic receptors are known to be involved in addictive processes[2], and have been suggested to be affected in schizophrenia [3, 4]. They have also been shown to be important for cognitive processes and memory. Nicotinic agonists are being developed as therapeutics for the treatment of Alzheimer's dementia [5]. Along with an increased understanding of the functional roles that nicotinic receptors may play in brain function has come an appreciation for the multiple receptor subtypes that exist in the brain. This proposal will extend our understanding of how a crucial physiological property, the permeability to divalent ions, is regulated both on the level of receptor subtype (i.e. subunit combination) and also in terms of the specific protein domains. Divalent ion permeability may be required for the neuronal plasticity associated with learning and memory and may also create a potential for excitotoxicity. We will express cloned nicotinic receptors in Xenopus oocytes, and with the study of chimeric and mutant subunits, we will identify the molecular elements that regulate calcium permeability, as well as the molecular elements that are involved with use-dependent inhibition of the receptors. By evaluating the relationships between the elements that regulate divalent ion permeability, and those which are associated with sensitivity to specific antagonists it may be possible to define therapeutics which may target specific functionally important receptor subtypes, either to spare those receptors important for cognitive processes, while targeting those involved in addictive processes, or to selectively block those which may put cells at risk of toxicity. The analysis of receptor physiology and pharmacology will be carried out both at the level of whole-cell currents and in terms of a detailed study of single channel properties. The study of a related series of bifunctional inhibitors will permit the disposition of inhibitory binding sites within the receptor complex to be evaluated. Our extensive preliminary data has provided us with a strong basis for experimental design in terms of approaches and candidate sequences for important structural domains. We have evidence that divalent ion permeability is regulated by the gamma subunits of muscle receptors and the alpha5 subunits of neuronal receptors. This model will be directly evaluated with antisense knockout experiments directed at eliminating the functional influences of these subunits in native receptors. The experiments in this proposal will provide important new insights into the nicotinic receptors of the brain in terms of their biophysical properties, their potential as therapeutic targets, and relationships between specific functional properties and drug sensitivity.