Mechanisms of Protein Regulation of Potassium Channels

描述（由申请人提供）：乙酰胆碱是一种与记忆和阿尔茨海默病有关的神经递质，结合并激活配体门控离子通道、G蛋白Gq偶联受体和G蛋白Gi偶联受体。因此，单一的神经递质可以进入神经元中的多种信号通路。因为这些通路中的每一个的激活都可能导致不同的生物反应，所以神经元必须在功能上分离这些不同的信号通路。神经元隔离信号通路的分子机制仍然是一个谜，也是当今神经科学家面临的最重要的问题之一。G蛋白门控内向整流钾通道（GIRK）的研究是一个很好的系统，用于确定受体特异性的分子决定因素。GIRK通道由G蛋白G β γ亚基打开，但在体外显示G β和G γ亚基的不同组合之间几乎没有区别。由于G β γ亚基的特定组合不能选择性地打开GIRK通道，因此理论上任何G蛋白偶联的神经递质受体都可以打开GIRK通道。然而，神经元确保乙酰胆碱在刺激G蛋白Gi偶联而不是Gq偶联的胆碱能受体后打开GIRK通道。这种类型的受体特异性在许多不同类型的神经递质信号通路中很常见。在这个提议中，将检查GIRK通道和G蛋白亚组之间的蛋白质-蛋白质相互作用决定受体偶联的特异性的假设。将使用生物化学检查直接蛋白质-蛋白质结合相互作用，电生理学评估GIRK通道的生理学，激光共聚焦显微镜可视化GIRK通道和神经递质受体的定位，最后荧光光谱测量活细胞中的蛋白质-蛋白质相互作用来测试该假设。此外，我们将使用海马神经元的培养物来鉴定GIRK通道中对靶向树突干很重要的结构域。这项资助的结果将大大推进我们对G蛋白偶联受体与GIRK通道偶联原理的理解，并可能有助于治疗由异常神经元膜兴奋性引起的人类疾病的特定药物策略。

DESCRIPTION (provided by applicant): Acetylcholine, a neurotransmitter implicated in memory and Alzheimer's disease, binds to and activates ligand-gated ion channels, G protein Gq-coupled receptors, and G protein Gi-coupled receptors. Hence, a single neurotransmitter can access a multitude of signaling pathways in neurons. Because activation of each of these pathways could result in a different biological response, neurons must functionally segregate these different signaling pathways. The molecular mechanism by which neurons isolate signaling pathways remains an enigma and is one of the most important questions facing Neuroscientists today. The study of G protein-gated inwardly rectifying K+ channels (GIRK) is an excellent system for identifying the molecular determinants of receptor specificity. GIRK channels are opened by the G protein G beta gamma subunits but show little discrimination among the different combinations of G beta and Ggamma, subunits in vitro. Because specific combinations of G beta gamma subunits do not selectively open GIRK channels, any G protein-coupled neurotransmitter receptor could theoretically open GIRK channels. However, neurons ensure that acetylcholine will open GIRK channels following stimulation of G protein Gi-coupled but not Gq-coupled cholinergic receptors. This type of receptor specificity is common among many different types of neurotransmitter signaling pathways. In this proposal, the hypothesis that a protein-protein interaction between the GIRK channel and a subset of G proteins determines the specificity of receptor coupling will be examined. The hypothesis will be tested using biochemistry for examining direct protein-protein binding interactions, electrophysiology for assessing the physiology of GIRK channels, laser confocal microscopy for visualizing the localization of GIRK channels and neurotransmitter receptors, and finally fluorescence spectroscopy for measuring protein-protein interactions in living cells. In addition, we will be using cultures of hippocampal neurons to identify the domains in GIRK channels that are important for targeting to the dendritic shafts. Results from this grant will significantly advance our understanding of the principles governing coupling of G protein-coupled receptors to GIRK channels, and may contribute to specific pharmaceutical strategies for treating humans with diseases that are caused by abnormal neuronal membrane excitability.