Synaptic function of BK channel-interacting proteins

BK 通道相互作用蛋白的突触功能

• 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
• 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

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Slo 1）几乎在体内普遍表达，具有许多重要的生物学功能。 生理功能，例如通过作用于神经元的突触前位点来调节神经递质释放。 该通道的突变可能导致多种疾病。Slo 1的生理功能在很大程度上依赖于 其在细胞膜中的表达水平以及与调节蛋白的相互作用。突变体的遗传筛选 抑制由C. elegans导致了两个鉴定 Slo 1体内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，Slo 1介导褪黑素的睡眠促进作用， Slo 1在调节神经递质释放和睡眠中生理作用取决于 褪黑激素分泌和褪黑激素受体的激活。在异源表达中，人Slo 1是 由褪黑素通过MT 1而不是MT 2褪黑素受体激活。然而， Slo 1在神经系统中的作用是调节蠕虫的睡眠，以及哺乳动物的Slo 1是否在天然的 神经元也可以通过特定的褪黑激素受体被褪黑激素激活。质谱分析 鉴定了与野生型相比，E3连接酶突变体中蛋白质大大增加。基因突变 编码这种蛋白质导致Slo 1功能增加，表明它是Slo 1的一种新的抑制性调节剂， E3连接酶通过促进这种假定的抑制性调节剂的降解来调节Slo 1。进一步研究 需要定义E3连接酶调节Slo 1的分子途径。该项目是 研究1）E3连接酶如何通过抑制性调节因子和其他蛋白质调节Slo 1; 2）在哪里和 Slo 1如何在神经系统中起作用以调节C中的睡眠。以及3）为什么MT 1而不是MT 2可以允许 在异源表达系统中褪黑激素对Slo 1的激活，以及褪黑激素是否也可以调节 Slo 1在小鼠脑中通过MT 1而不是MT 2。我们将使用以下组合来回答这些问题： 电生理学、遗传学、细胞学和分子生物学方法。拟议研究的结果如下： 有望产生关于Slo 1如何与其他蛋白质相互作用以调节细胞增殖的重要新知识。 兴奋性神经递质释放和行为