Measurement of GPCR-mediated thallium flux:GIRK (RMI)

描述（由申请人提供）：靶向G蛋白偶联受体和离子通道的药理学药物代表了跨越多种人类疾病的有前景的临床药物。我们已经开发了一种筛选策略，通过测量铊通量通过G蛋白调节的抑制整流钾（K+）（GIRK）通道的Gi/o连接的受体。使用这种技术在HEK细胞稳定表达GIRK 1和2通道，我们已经产生了初步的数据，使用毒蕈碱和肾上腺素能激动剂和拮抗剂，并观察到剂量依赖性调节这些通道的内源性M2/M4受体和？2C肾上腺素能受体选择性药物。我们已经将该测定法适应于384孔板形式，并且已经进行了初始Z'实验，其表明该测定法适合HTS。我们建议进行高通量筛选新的激动剂在？2C受体;在这些研究的过程中，预计我们将确定新的激动剂GIRK 1/2通道以及。很少有具体的药理学工具存在针对？2C受体，并且由于其在精神疾病和正常心脏功能中的拟议作用，预计针对该受体的激动剂将提供关键的测试工具？2C受体在体内的功能。已知GIRK 1/2通道参与神经元兴奋性，并且已经提出该受体的激动剂可以为癫痫和疼痛提供新的治疗途径。概述：G蛋白偶联受体（GPCR）和离子通道代表了人类疾病中可获得的治疗靶点。我们已经开发了一种新的检测方法，适合高通量的策略，它可以用来筛选大型的化学库中的化合物从受体到离子通道的信号转导通路内的作用。我们建议使用这种检测来确定化合物相互作用的？2C肾上腺素能受体以及在我们的测定系统中使用的直接调节钾通道的试剂。确定的化合物将提供新的药理学工具来测试的作用？2C肾上腺素能受体在精神和心血管疾病中的作用以及GIRK 1/2通道在神经元兴奋性调节中的作用

DESCRIPTION (provided by applicant): Pharmacological agents targeting G-protein coupled receptors and ion channels represent promising clinical agents spanning a multitude of human diseases. We have developed a screening strategy for Gi/o-linked receptors by measuring thallium flux through G protein regulated Inwardly Rectifying Potassium (K+) (GIRK) channels. Using this technique in HEK cells stably expressing GIRK 1 and 2 channels, we have generated preliminary data using muscarinic and adrenergic agonists and antagonists and have observed dose dependent regulation of these channels by endogenous M2/M4 receptors and ?2C adrenergic receptor selective agents. We have adapted this assay to a 384 well plate format and have performed initial Z' experiments that indicate that the assay is amenable to HTS. We propose to conduct a high throughput screen for novel agonists at the ?2C receptor; in the course of these studies, it is anticipated that we will identify novel agonists of GIRK 1/2 channels as well. Very few specific pharmacological tools exist targeting the ?2C receptor and due to its proposed role in psychiatric disorders and normal cardiac function it is anticipated that agonists targeting this receptor would provide critical tools to test ?2C receptor function in vivo. GIRK1/2 channels are known to be involved in neuronal excitability and it has been proposed that agonists for this receptor may provide new therapeutic avenues for epilepsy and pain. Lay summary: G-protein coupled receptors (GPCRs) and ion channels represent accessible therapeutic targets in human disease. We have developed a new assay, amenable to high throughput strategies, which can be used to screen large chemical libraries for compounds acting within the signal transduction pathway from receptor to ion channel. We propose to use this assay to identify compounds interacting with the ?2C adrenergic receptor as well as agents directly modulating the potassium channel employed in our assay system. Identified compounds will provide new pharmacological tools to test the role of the ?2C adrenergic receptor in psychiatric and cardiovascular disorders and the role of GIRK 1/2 channels in the modulation of neuronal excitability.