Regulation of Neuronal Nicotinic Acetylcholine receptors

描述（由申请人提供）：本研究的目的是了解控制脊椎动物胆碱能神经递质系统发育的转录机制。脊椎动物基因的一个保守簇，顺序为β 4、α 3和α 5，编码在外周和中枢神经元中组装成几种神经元烟碱乙酰胆碱受体（nAchR）亚型的亚基。由所有三种亚基组成的一种亚型对于肾上腺嗜铬细胞中以及交感神经和副交感神经系统的节前和节后神经元之间的兴奋性突触后传递是必不可少的。含有β 4和α 3亚基的亚型对于视网膜中的胆碱能突触传递很重要。在这些亚型组装之前，编码β 4、α 3和α 5的基因必须在正确的时间在适当的神经元中转录。然而，控制亚基表达的转录机制知之甚少。我们已经在簇中鉴定出一个增强子（beta43 '），它可能对神经元中亚基基因的转录很重要。我们还表明ETS结构域因子相互作用对β 43 '的神经元活性很重要。在该提议中，我们希望确定β 43 '的生物学相关性及其与ETS因子的相互作用，用于组装由β 4、α 3和α 5制成的不同受体亚型。我们的假设是β 43 '通过ETS相互作用控制成簇基因的神经元特异性转录。我们已经准备了转基因小鼠表达的每一个集群的基因从PI-细菌人工染色体（PAC）。我们将准备额外的转基因小鼠品系，这些小鼠在增强子ETS结合位点携带突变。然后，将在表达簇的各种神经元细胞类型中研究每个簇基因的转录。我们还将通过研究我们的Pet-1敲除小鼠中的亚基RNA和蛋白质水平来研究该簇是否是Pet-1 ETS因子的转录靶点。我们还将研究ETS因子在表达该簇的神经元细胞类型中的表达。本研究将进一步加深我们对神经元胆碱能系统发育和特定nAchR亚型组装机制的理解。这也将有助于揭示ETS因子在脊椎动物神经元中的不明确功能。

DESCRIPTION (provided by applicant): The objective of the research proposed here is to understand transcriptional mechanisms that control cholinergic neurotransmitter system development in vertebrates. A conserved cluster of vertebrate genes ordered beta4, alpha3, and alpha5 encode subunits that are assembled into several neuronal nicotinic acetylcholine receptor (nAchR) subtypes in peripheral and central neurons. One subtype composed of all three subunits is essential for excitatory post-synaptic transmission in adrenal chromaffin cells and between pre- and post-ganglionic neurons of the sympathetic and parasympathetic nervous systems. Subtypes containing beta4 and alpha3 subunits are important for cholinergic synaptic transmission in retina. Before these subtypes can be assembled the genes encoding beta4, alpha3, and alpha5 must be transcribed in appropriate neurons at the correct time. However, the transcriptional mechanisms that control subunit expression are poorly understood. We have identified an enhancer (beta43') within the cluster that is likely to be important for transcription of the subunit genes in neurons. We also showed that ETS domain factor interactions are important for neuronal activity of beta43'. In this proposal we wish to determine the biological relevance of beta43' and its interactions with ETS factors for the assembly of different receptor subtypes made from beta4, alpha3, and alpha5. Our hypothesis is that beta43' controls neuron specific transcription of the clustered genes through ETS interactions. We have prepared transgenic mice expressing each of the clustered genes from a Pl-bacterial artificial chromosome (PAC). We will prepare additional lines of transgenic mice that carry mutations in the enhancer ETS binding sites. Then transcription of each clustered gene will be investigated in various neuronal cell types that express the cluster. We will also investigate whether the cluster is a transcriptional target of the Pet-1 ETS factor by investigating subunit RNA and protein levels in our Pet-1 knock out mice. We will also investigate ETS factor expression in neuronal cell types that express the cluster. This research will further our understanding of the mechanisms governing the development of neuronal cholinergic systems and assembly of specific nAchR subtypes. It will also help to reveal the poorly defined functions of ETS factors in vertebrate neurons.