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

描述(由申请人提供)：针对G蛋白偶联受体和离子通道的药理学药物代表着跨越多种人类疾病的有前途的临床药物。我们开发了一种通过G蛋白调节的内向整流钾(GIRK)(GIRK)通道测量铊的流量来筛选Gi/o连锁受体的策略。在稳定表达GIRK 1和GIRK 2通道的HEK细胞中，我们用M受体和肾上腺素能激动剂和拮抗剂产生了初步数据，并观察到内源性M2/M4受体和？2C肾上腺素能受体选择剂对这些通道的剂量依赖性调节。我们已经将这一检测方法适应于384孔板格式，并进行了初步的Z‘实验，表明该检测方法是服从HTS的。我们建议对？2C受体上的新激动剂进行高通量筛选；在这些研究过程中，我们预计也将识别GIRK 1/2通道的新激动剂。目前针对？2C受体的特定药理工具很少，由于其在精神疾病和正常心脏功能中的作用，预期靶向该受体的激动剂将提供关键的工具来测试体内的？2C受体功能。已知GIRK1/2通道与神经元兴奋性有关，已提出该受体的激动剂可能为癫痫和疼痛提供新的治疗途径。综述：G蛋白偶联受体(GPCRs)和离子通道在人类疾病中是可获得的治疗靶点。我们已经开发了一种新的分析方法，适用于高通量策略，可以用来筛选大的化学库，寻找从受体到离子通道的信号转导途径中作用的化合物。我们建议使用这种方法来鉴定与？2C肾上腺素能受体相互作用的化合物，以及在我们的检测系统中使用的直接调节钾通道的药物。已确定的化合物将提供新的药理学工具来测试？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.