MOLECULAR ACTIONS OF VOLATILE ANESTHETICS ON G-PROTEINS

挥发性麻醉剂对 G 蛋白的分子作用

• 批准号: 6031605
• 负责人: Mario J Rebecchi
• 金额: $ 28.49万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6031605
• 关键词: G protein; GABA receptor; Sf9 cell line; anesthetics; biological signal transduction; calcium flux; cell membrane; fluorescence resonance energy transfer; gene expression; guanine nucleotide exchange factors; guanosine diphosphate; guanosine triphosphate; inositol phosphates; magnesium ion; microcalorimetry; pharmacokinetics; protein protein interaction; protein structure function; receptor coupling; stoichiometry

DESCRIPTION (adapted from applicant's abstract): Volatile anesthetics perturb the coupling of metabotropic receptors to specific heterotrimeric guanine nucleotide-binding proteins (G-proteins), but the relevance to anesthesia and its molecular basis are not understood. Heterotrimeric G-proteins, consisting of alpha, beta, and gamma subunits, are GTP-driven molecular switches that couple metabotropic receptors to downstream effectors, including ion channels. In the inactive state, the G-protein is a heterotrimer with GDP tightly bound to the Galpha subunit. Binding of agonist to the receptor triggers exchange of GTP for bound GDP, hydrolyzing the former in the presence of Mg2+. The GTP-charged alpha subunit dissociates from betagamma and may be free to diffuse some distance on the membrane surface. Either the alpha subunit bound to GTP or the free betagamma heterodimer or both regulate effector enzymes such adenyl cyclase (AC) and phospholipase C, or modulate ion channels, thereby amplifying the initial receptor stimulus. Thus, alpha subunits function as on/off switches based on the occupant of the nucleotide binding site, GTP or GDP, such that alterations in nucleotide exchange, like those induced by volatile anesthetics, modulate signal output. To test for correlation between clinical potency and inhibition of GDP/GTP exchange, various anesthetic compounds, as well as non-immobilizing drugs, will be tested on Galpha subunits. Similar experiments will be performed on living cells in which the pathways are reconstructed by expression of the signaling components: receptors, G-protein subunits and effectors. The hypothesis that volatile agents disrupt receptor/effector coupling by locking the Galpha subunit into a complex with Gbetagamma will be tested on artificial membranes by measuring the lateral interactions of these proteins using fluorescence resonance energy transfer. To understand the molecular basis of the anesthetic effect, intrinsic fluorescence and isothermal titration microcalorimetry will be used to measure the binding of these drugs to soluble Galpha subunits. Finally, regions essential to the actions of anesthetics will be established by swapping homologous portions of sensitive and insensitive alpha subunits. These studies will generate predictions for future experiments to be conducted on animals exposed to anesthetic agents. Completion of the proposed study will also form the basis for future crystallographic work on the nature of interactions between anesthetics and these potential protein targets.

描述（改编自申请人的摘要）：挥发性麻醉药扰动 代谢受体与特定异三聚体鸟嘌呤的偶联 核苷酸结合蛋白（G蛋白），但与麻醉和 它的分子基础尚不清楚。异三聚体G蛋白，组成 Alpha，Beta和Gamma亚基是GTP驱动的分子开关 夫妇代谢受体，包括包括离子通道在内的下游效应子。 在不活跃的状态下，G蛋白是具有GDP紧密结合的异构体 到Galpha亚基。激动剂与受体触发的结合交换 GDP的GTP，在存在Mg2+的情况下水解前者。这 与betagamma的GTP充电亚基分离物，可以自由扩散 膜表面的一些距离。绑定到GTP的Alpha亚基或 游离的betagamma异二聚体或两者都调节效应酶此类腺苷 环酶（AC）和磷脂酶C或调节离子通道，从而扩增 初始受体刺激。因此，alpha亚基如开/关开关的功能 基于核苷酸结合位点GTP或GDP的占用者，这样 核苷酸交换的改变，例如挥发性麻醉剂引起的核苷酸交换， 调节信号输出。测试临床效力与 抑制GDP/GTP交换，各种麻醉化合物以及 非弹性药物将在Galpha亚基上进行测试。类似的实验 将对途径重构的活细胞进行 信号成分的表达：受体，G蛋白亚基和 效应子。挥发性剂破坏受体/效应子的假设 通过将Galpha亚基锁定到与Gbetagamma的建筑群中，将 通过测量这些的横向相互作用在人造膜上测试 使用荧光共振能传递的蛋白质。理解 麻醉作用，内在荧光和等温的分子基础 滴定微量钙化法将用于测量这些药物的结合 用于可溶的galpha亚基。最后，地区对行动至关重要 通过交换敏感的同源部分来建立麻醉药 和不敏感的α亚基。这些研究将为 未来对暴露于麻醉剂的动物进行的实验。 拟议研究的完成还将构成未来的基础 关于麻醉和之间相互作用的性质的晶体学工作 这些潜在的蛋白质靶标。