Determination of the GIRK channel proteome

G蛋白门控的内向整流钾(GIRK)通道的激活提供了关键的抑制 大脑中减少神经元放电的信号。奖赏途径中的许多神经递质，如 多巴胺和GABA通过GIRK通道与GPCRs偶联。先前的研究已经 确定了GIRK通道在对精神刺激剂的反应中的作用。一个复杂的蛋白质网络 被假设为支持毒品依赖的变化，贩运和瞄准GIRK渠道以 突触后抑制性突触，但这些蛋白的特性在很大程度上仍不清楚。致信地址 这一领域的重大空白，是一种基于邻近关系的生物素识别创新技术(简称 AS iBio-ID)将用于鉴定GIRK通道蛋白质组中的新蛋白质(目标1)。在这份提案中， GIRK通道将被设计成在体内(在通道的50纳米范围内)生物素化蛋白质， 然后，生物素化的蛋白质将被提纯，并用串联质谱仪进行鉴定。初步 数据显示，与生物素基化酶融合的GIRK通道是有功能的，并且可以生物素化 活体中的蛋白质，导致鉴定可能的GIRK通道调节因子。以下是量化 分析，候选GIRK通道调节蛋白将被评估与功能相互作用 神经元中的GIRK通道。可卡因作用下GIRK通道蛋白质组的变化 还将研究作为成瘾模型的敏感化(目标2)。这些实验将为 首次全面列出了GIRK通道中的蛋白质组，并导致了新的身份 GIRK通道的药物依赖性调节因子。奖赏通路中新蛋白质靶点的发现 可能会导致新的成瘾治疗方法的开发。

Activation of G protein-gated inwardly rectifying potassium (GIRK) channels provides a key inhibitory signal in the brain that reduces neuronal firing. Many neurotransmitters in the reward pathway, such as dopamine and GABA, couple to GPCRs that signal through GIRK channels. Prior studies have established a role for GIRK channels in the response to psychostimulants. A complex network of proteins is hypothesized to support drug-dependent changes in trafficking and targeting of GIRK channels to postsynaptic inhibitory synapses, but the identity of these proteins remain largely unknown. To address this major gap in the field, an innovative technique of proximity-dependent biotin identification (referred to as iBio-ID) will be used to identify new proteins in the GIRK channel proteome (Aim 1). In this proposal, GIRK channels will be engineered to biotinylate proteins in vivo (within 50 nanometers of the channel), and then biotinylated proteins will be purified and identified using tandem mass spectrometry. Preliminary data show that GIRK channels fused to the biotinylating enzyme are functional, and can biotinylate proteins in vivo, leading to identification of putative GIRK channel regulators. Following quantitative analyses, candidate GIRK channel regulator proteins will be assessed for functional interaction with GIRK channels in neurons. Changes in the GIRK channel proteome with cocaine, using the locomotor sensitization as a model of addiction, will also be studied (Aim 2). These experiments will provide for the first time a comprehensive list of proteins in the GIRK channel proteome, and lead to the identity of new drug-dependent regulators of GIRK channels. Discovery of novel protein targets in the reward pathway could lead to the development of new treatments for addiction.