Signaling pathways that modulate neuronal activity

项目摘要 神经元的活动可以通过与异源三聚体G proteins.这些G蛋白信号转导通路的激活可能导致离子通道的调节 在细胞水平上改变神经元的兴奋性，最终导致生物体行为的改变。 水平我们工作的长期目标是识别和理解神经调节的机制基础， 途径。这项提案的目标是确定新的调节途径，导致调制的 特定离子通道，NALCN/NCA通道。NALCN/NCA离子通道是一种公认的阳离子通道 与电压门控钠和钙通道有关，但其精确的细胞作用和调节不 很好理解。然而，NALCN或其相关亚基中的突变与人类免疫缺陷直接相关。 以一系列症状为特征的神经系统疾病，包括运动和肌肉异常 宫缩、智力残疾和癫痫发作。此外，模式生物中该通道的突变 引起强烈的神经元表型，包括节律行为和神经元兴奋性的缺陷， 证明了这个通道的生理重要性。通过线虫的正向遗传筛选 C.我们发现NCA离子通道被一条新的信号转导途径激活， 异源三聚体G蛋白Gq的下游。激活的Gq直接结合并刺激鸟嘌呤 Trio RhoGEF的核苷酸交换活性激活小G蛋白Rho，导致调节 通过未知的机制破坏了NCA频道在这里，我们将确定Gq-Rho信号如何调节 NCA活性通过研究在我们的屏幕中确定的其他因素。在目标1中，我们将重点关注G蛋白- 偶联受体激酶GRK-2。我们的遗传数据支持GRK-2调节多巴胺的假设 通过Gq-Rho途径负调节NCA活性的信号传导。我们将进行基因， 生物化学，细胞成像和电生理学实验，以确定GRK-2如何与 影响多巴胺受体的活性，最终调节NCA通道的活性。在目标2中， 将集中在丝裂原活化蛋白激酶（MAPK）途径，调节Gq-Rho激活NCA。 我们将确定该信号通路的成员，并确定它们如何调节Gq的输出， Rho-NCA途径。这项工作很有意义，因为它将确定信号通路， 通过生理和医学上重要的离子通道调节神经元活性。拟议的工作是 创新，因为它将填补我们对NALCN/NCA离子通道如何激活的理解的空白， 确定这一渠道的新的调节机制。

Project summary Neuronal activity can be modulated by transmitters that act through receptors coupled to heterotrimeric G proteins. Activation of these G protein signal transduction pathways may lead to the modulation of ion channels that alter neuronal excitability at the cellular level, ultimately causing changes in behavior at the organismal level. The long-term goal of our work is to identify and understand the mechanistic basis of neuromodulatory pathways. The goal of this proposal is to identify new regulatory pathways that lead to the modulation of a specific ion channel, the NALCN/NCA channel. The NALCN/NCA ion channel is a putative cation channel related to voltage-gated sodium and calcium channels, but whose precise cellular role and regulation are not well understood. However, mutations in NALCN or its associated subunits have been directly linked to human neurological diseases characterized by a range of symptoms, including abnormal movements and muscle contractions, intellectual disability, and seizures. Additionally, mutations in this channel in model organisms cause strong neuronal phenotypes including defects in rhythmic behaviors and neuronal excitability, demonstrating the physiological importance of this channel. Through a forward genetic screen in the nematode C. elegans, we found that the NCA ion channel is activated by a new signal transduction pathway acting downstream of the heterotrimeric G protein Gq. Activated Gq directly binds and stimulates the guanine nucleotide exchange activity of the Trio RhoGEF to activate the small G protein Rho, leading to the modulation of the NCA channel through unknown mechanisms. Here we will determine how Gq-Rho signaling modulates NCA activity by studying additional factors identified in our screen. In Aim 1, we will focus on the G protein- coupled receptor kinase GRK-2. Our genetic data support the hypothesis that GRK-2 modulates dopamine signaling that negatively regulates NCA activity through the Gq-Rho pathway. We will perform genetic, biochemical, cellular imaging, and electrophysiological experiments to determine how GRK-2 interacts with and affects the activity of dopamine receptors to eventually modulate the activity of the NCA channels. In Aim 2, we will focus on a mitogen-activated protein kinase (MAPK) pathway that modulates Gq-Rho activation of NCA. We will identify the members of this signaling pathway and determine how they modulate output of the Gq- Rho-NCA pathway. The proposed work is significant because it will identify the signaling pathways that modulate neuronal activity via a physiologically and medically important ion channel. The proposed work is innovative because it will close gaps in our understanding of how the NALCN/NCA ion channel is activated and identify new mechanisms of regulation of this channel.