Determination of the GIRK channel proteome

G蛋白门控内向整流钾（GIRK）通道的激活提供了一个关键的抑制性信号通路。 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与通过GIRK通道发出信号的GPCR偶联。先前的研究 GIRK通道在对精神兴奋剂的反应中发挥作用。蛋白质的复杂网络 假设支持GIRK通道的贩运和靶向的药物依赖性变化， 突触后抑制性突触，但这些蛋白质的身份仍然在很大程度上未知。解决 该领域中的这一主要空白是一种邻近依赖性生物素鉴定的创新技术（参见 作为iBio-ID）将用于鉴定GIRK通道蛋白质组（Aim 1）中的新蛋白质。在这一提议中， GIRK通道将被设计成在体内生物素化蛋白质（在通道的50纳米内）， 然后使用串联质谱法纯化和鉴定生物素化的蛋白质。初步 数据显示，与生物素化酶融合的GIRK通道是功能性的， 蛋白质在体内，导致推定的GIRK通道调节剂的鉴定。以下定量 分析中，将评估候选GIRK通道调节蛋白的功能相互作用， 神经元中的GIRK通道。可卡因引起的GIRK通道蛋白质组的变化，使用运动 致敏作为成瘾模型，也将进行研究（目标2）。这些实验将为 第一次在GIRK通道蛋白质组的蛋白质的全面列表，并导致新的身份 GIRK通道的药物依赖性调节剂。奖赏途径中新的蛋白质靶点的发现 可能会导致新的成瘾治疗方法的发展。

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.