Synaptic function of BK channel-interacting proteins

BK 通道相互作用蛋白的突触功能

基本信息

批准号：
10590677
负责人：
ZHAO-WEN WANG
金额：
$ 55.17万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-12-01 至 2026-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10590677
关键词：
Action Potentials Alcohol dependence Amino Acids Ataxia Behavior Behavioral Binding Biochemical Biological Biological Assay Brain CBA/CaJ Mouse Caenorhabditis elegans Cell Communication Cell membrane Cells Chemical Agents Chimera organism Clathrin Co-Immunoprecipitations Complement Disease Dominant-Negative Mutation Electrophysiology (science)Endocytosis Epilepsy Fluorescence Functional disorder Gene Mutation Genetic Genetic Screening Guanine Nucleotide Exchange Factors Guanosine Human Hyperactivity Immunohistochemistry In Vitro Intellectual functioning disability Knock-out Knockout Mice Knowledge Ligase Ligation Lysine Mass Spectrum Analysis Mediating Melatonin Melatonin Receptors Modeling Molecular Monomeric GTP-Binding Proteins Mus Mutate Mutation Nervous System Neurons Nucleotides Pathway interactions Phenotype Physiologic pulse Physiological Potassium Channel Property Proteins Regulation Ring Finger Domain Role Site Sleep Slice Source Surface Synaptic Transmission System Testing Ubiquitination Xenopus oocyte channel blockers experimental study forward genetics genetic approach genetic regulatory protein in vivo insight knock-down large-conductance calcium-activated potassium channels loss of function mutant nervous system disorder neural circuit neuronal excitability neurotransmitter release novel overexpression paxilline postsynaptic presynaptic sleep behavior suprachiasmatic nucleus synaptic function ubiquitin-protein ligase

项目摘要

The BK channel (also known as Slo1) is almost ubiquitously expressed in the body with many important physiological functions, such as regulating neurotransmitter release by acting at presynaptic sites of neurons. Mutations of the channel may cause diverse diseases. Physiological functions of Slo1 depend to great degrees on its expression level in the cell membrane and interactions with regulatory proteins. Genetic screen for mutants that suppress a sluggish phenotype caused by a hyperactive Slo1 in C. elegans led to the identification of two proteins required for Slo1 physiological functions in vivo, including a melatonin receptor and an ubiquitin E3 ligase. Electrophysiological and behavioral analyses indicate that Slo1 mediates melatonin’s sleep-promoting effect in worms, and that Slo1’s physiological roles in regulating neurotransmitter release and sleep depend on melatonin secretion and activation of the melatonin receptor. In a heterologous expression, human Slo1 is activated by melatonin through the MT1 but not MT2 melatonin receptor. However, it remains to be determined where Slo1 acts in the nervous system to regulate sleep in worms, and whether mammalian Slo1 in native neurons may be also activated by melatonin through a specific melatonin receptor. Mass spectrometry analyses identified a protein greatly increased in mutants of the E3 ligase compared with wild type. Mutations of the gene encoding this protein led to increased Slo1 function, suggesting that it is a novel inhibitory regulator of Slo1, and that the E3 ligase regulates Slo1 by facilitating degradation of this putative inhibitory regulator. Further studies are needed to define a molecular pathway through which the E3 ligase regulates Slo1. This project is to investigate 1) how the E3 ligase regulates Slo1 through the inhibitory regulator and other proteins; 2) where and how Slo1 acts in the nervous system to regulate sleep in C. elegans; and 3) why MT1 but not MT2 may allow Slo1 activation by melatonin in the heterologous expression system, and whether melatonin can also regulate Slo1 in mouse brain through MT1 but not MT2. We will answer these questions using a combination of electrophysiological, genetic, cellular, and molecular biological approaches. Results of the proposed studies are expected to produce important new knowledge about how Slo1 interacts with other proteins to regulate cellular excitability, neurotransmitter release, and behavior.

BK通道（也称为SLO1）几乎在体内表达了许多重要的生理功能，例如通过在神经元的突触前作用来调节神经递质释放。通道的突变可能会导致多种疾病。 SLO1的生理功能在很大程度上取决于在其在细胞膜中的表达水平以及与调节蛋白的相互作用。突变体的遗传筛查这抑制了由秀丽隐杆线虫中多活跃Slo1引起的缓慢表型，导致了两个体内SLO1生理功能所需的蛋白质，包括褪黑激素受体和泛素E3 连接酶。电生理和行为分析表明SLO1介导褪黑激素的睡眠促进在蠕虫中的影响，以及SLO1在控制神经递质释放和睡眠中的身体作用取决于褪黑激素分泌和褪黑激素受体的激活。在异源表达中，人类slo1是通过MT1而不是MT2褪黑激素受体激活。但是，这尚待确定 SLO1在神经系统中作用以调节蠕虫的睡眠，以及哺乳动物Slo1是否在本地神经元也可以通过特定的褪黑激素受体激活神经元。质谱分析与野生型相比，E3连接酶突变体的蛋白质大大增加。基因的突变编码这种蛋白质导致SLO1功能增加，表明它是一种新型的抑制性调节剂，并且 E3连接酶通过支持该推定的抑制性调节剂的降解来调节SLO1。进一步的研究需要定义E3连接酶调节SLO1的分子途径。这个项目是研究1）E3连接酶如何通过抑制性调节剂和其他蛋白质调节SLO1； 2）在哪里和 SLO1在神经系统中的作用如何调节秀丽隐杆线虫的睡眠； 3）为什么MT1而不是MT2可能允许褪黑激素在异源表达系统中激活，以及褪黑激素是否也可以调节通过MT1而非MT2中的小鼠大脑中的SLO1。我们将使用的结合来回答这些问题电生理，遗传，细胞和分子生物学方法。拟议研究的结果是预计会产生有关SLO1如何与其他蛋白质相互作用以调节细胞的重要新知识兴奋性，神经递质释放和行为。