MOLECULAR MECHANISMS OF OLFACTORY INTEGRATION

嗅觉整合的分子机制

• 批准号: 2127674
• 负责人: Timothy S McClintock
• 金额: $ 8.91万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2127674
• 关键词: G protein; Malacostraca; Xenopus; Xenopus oocyte; adenylate cyclase; biological signal transduction; chemoreceptors; cyclic AMP; dendrites; electrophysiology; immunoprecipitation; in situ hybridization; inositol phosphates; laboratory rabbit; membrane channels; molecular cloning; neural information processing; northern blottings; olfactions; phospholipase C; phosphorylation; potassium channel; receptor coupling; second messengers; western blottings

Odorant mixtures which simultaneously activate both the adenylate cyclase and phospholipase C transduction pathways in individual lobster olfactory receptor neurons cause an integrated cellular response. Co-activation of interacting transduction pathways may increase the quality coding capacity of the receptor neuron population by increasing the diversity of receptor neuron response types. The objective of this proposal is test this broad hypothesis by fully characterizing these two transduction pathways and their interactions. Experiments using molecular cloning, biochemical, and electrophysiological techniques are proposed. The hypotheses to be tested are: 1) that all receptor neurons possess both transduction pathways, 2) that cDNA clones from G protein alpha subunits, effector enzymes and second messenger-gated ion channels participate in the series of molecular interactions necessary to mediate olfactory transduction and desensitization, and 3) that any of several biochemical mechanisms mediate interactions between the transduction pathways. Lobster olfactory cDNA sequences for all or part of two G protein alpha subunits and a phospholipase C have been cloned using homology cloning techniques, which will also be used to clone an olfactory adenylate cyclase and a cyclic nucleotide-gated potassium channel. An IP3-gated channel cDNA clone will be isolated by immunoscreening of an expression library. In situ hybridizations will determine the distribution of each clone (hypothesis 1). Antibodies raised to each sequence will be used to test the presence of each clone in olfactory dendrites (hypothesis 2). The interaction pathway from G-protein to enzyme to channel will be studied in heterologous expression studies in eukaryotic cells and by co-immunoprecipitation from olfactory cilia (hypothesis 2). The effects of calcium, calmodulin, and phosphorylation upon the cloned enzymes and ion channels will be determined in functional expression studies (hypotheses 2 and 3).

同时激活腺苷酸环化酶的气味混合物 单个龙虾嗅觉中的磷脂酶C转导途径 受体神经元引起整合的细胞反应。 共同激活 相互作用的转导途径可能会增加质量编码能力 通过增加受体的多样性，受体神经元种群的多样性 神经元反应类型。 该提议的目的是测试这一广泛 通过充分表征这两个转导途径和 他们的互动。 使用分子克隆，生化和 提出了电生理技术。 要测试的假设 是：1）所有受体神经元都具有两个转导途径，2） G蛋白α亚基，效应酶和第二个cDNA克隆 信使门控离子通道参与了一系列分子 介导嗅觉转导和 脱敏，3）几种生化机制中的任何一个都介导 转导途径之间的相互作用。 龙虾嗅觉cDNA 两个G蛋白α亚基和A的全部或一部分的序列 磷脂酶C已使用同源克隆技术克隆到克隆中， 还将用于克隆嗅觉腺苷酸环化酶和环状 核苷酸门控钾通道。 IP3门控通道cDNA克隆将 通过对表达式文库进行免疫统计来隔离。 原位 杂交将确定每个克隆的分布（假设 1）。 对每个序列提出的抗体将用于测试存在 嗅树突中的每个克隆（假设2）。 相互作用 从G蛋白到酶再到通道的途径将在异源中研究 在真核细胞和通过共免疫沉淀中的表达研究 嗅觉纤毛（假设2）。 钙，钙调蛋白和 将确定克隆酶和离子通道上的磷酸化 在功能表达研究中（假设2和3）。