Synaptic function of BK channel-interacting proteins

BK通道(也称为slo1)在体内几乎无处不在地表达，有许多重要的 生理功能，如通过作用于神经元的突触前部位来调节神经递质的释放。 该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依赖于 它在细胞膜上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 抑制线虫中由高度活跃的slo1引起的迟缓表型导致了两个 SL1体内生理功能所需的蛋白质，包括褪黑素受体和泛素E3 连接酶。电生理和行为学分析表明，slo1介导了褪黑素的促眠作用 S在调节神经递质释放和睡眠中的生理作用依赖于 褪黑素的分泌和褪黑素受体的激活。在异源表达中，人类slo1是 由褪黑素通过MT1而不是MT2褪黑素受体激活。然而，这一点还有待确定。 Slo1在神经系统中起作用来调节蠕虫的睡眠，以及哺乳动物slo1是否为天然的 神经元也可以通过特定的褪黑素受体被褪黑素激活。质谱分析 与野生型相比，E3连接酶突变体中的一种蛋白质大大增加。基因突变 编码该蛋白导致了slo1功能的增强，表明它是slo1的一个新的抑制调节因子，并且 E3连接酶通过促进这一假定的抑制调节因子的降解来调节sL1。进一步研究 是确定E3连接酶调节slo1的分子途径所必需的。这个项目是为了 研究1)E3连接酶如何通过抑制调节因子和其他蛋白质调节sL1；2)在哪里和 Slo1如何在神经系统中调节线虫的睡眠；以及3)为什么MT1而不是MT2可能允许 褪黑素在异源表达系统中对SLO1的激活及其是否也具有调节作用 SLO1通过MT1进入小鼠脑内，而不通过MT2进入小鼠脑。我们将结合以下几个方面回答这些问题 电生理、遗传、细胞和分子生物学方法。建议的研究结果如下 有望产生关于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。

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.