Mechanisms of GIRK channel activation by the antiepileptic compound ML297.

抗癫痫化合物 ML297 激活 GIRK 通道的机制。

• 批准号: 8783819
• 负责人: Nidaa Othman Marsh
• 金额: $ 1.58万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2014-08-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8783819
• 关键词: Address; Adverse effects; Affect; Agonist; Alcohols; American; Antiepileptic Agents; Anxiety; Binding; Binding Sites; Biological Assay; Brain; Butyric Acids; Calcium Channel; Cells; Chemicals; Chemosensitization; Data; Development; Diagnosis; Disease; Disease remission; Drug Targeting; Effectiveness; Electrophysiology (science); Epilepsy; Family; Future; G protein-coupled inwardly-rectifying potassium channel; GIRK1 subunit, G protein-coupled inwardly-rectifying potassium channel; GIRK2 subunit, G protein-coupled inwardly-rectifying potassium channel; GIRK3 subunit, G protein-coupled inwardly-rectifying potassium channel; GTP-Binding Proteins; Gated Ion Channel; Glutamate Receptor; Goals; Heart; Individual; Ion Channel; Kinetics; Life; Ligands; Location; Mediating; Methods; Modification; Molecular Biology; Mutate; Mutation; Neuraxis; Parkinson Disease; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Play; Point Mutation; Potassium; Potassium Channel; Quality of life; Role; Sedation procedure; Seizures; Series; Site; Site-Directed Mutagenesis; Sodium Channel; Testing; Therapeutic; Time; Training; Treatment Protocols; Work; addiction; base; design; experience; mutant; nervous system disorder; neuronal excitability; neurotransmission; novel; patch clamp; public health relevance; research study; stoichiometry; tool; voltage

DESCRIPTION (provided by applicant): Epilepsy affects nearly 3 million Americans, with 10 percent of all new patients failing to gain control of their seizures with correct medicinal management. Current anti-epileptic therapies restore normal neurotransmission by targeting ion channels in the brain. Many individuals develop tolerance to their prescriptions, experience side effects and require continuous modification of their treatment regimen over time. Today, there is an inherent need for anti-epileptic drug therapies that can reduce these side effects and maintain effectiveness over longer periods of time. G-protein inwardly rectifying potassium (GIRK) channels regulate neurotransmission in the brain. Due to their regional distribution throughout the brain, they have been implicated in many neurological disorders including addiction, anxiety and epilepsy and Parkinson's disease. Recent studies have demonstrated that GIRK channels can be directly activated by alcohol through an isolated binding pocket formed by the N- terminus, ¿ D- ¿ E and ¿ L- ¿ M. This binding site, which lies at the interface between adjacent subunits, could be a target for other ligands. ML297, an anti-epileptic compound, was recently identified and released as a tool to study GIRK channel function. Both the mechanism by which ML297 activates GIRK channels and the binding site on the channel are not known. Isolating this mechanism could be used to develop better anti-epileptic therapies in the future. We hypothesize that ML297 induces channel activation through direct binding to the alcohol binding site. Preliminary studies have shown that ML297 is inactive at GIRK channels lacking the GIRK1 subunit. In this proposal, I have designed a series of experiments to isolate the binding site for ML297 at different GIRK channels through patch clamp electrophysiology and site-directed mutagenesis under three aims. For aims 1 and 2, I will use whole-cell patch clamp electrophysiology to examine macroscopic changes in gating induced by ML297. In aim 1, I will establish a mechanism for ML297 binding to the GIRK1 subunit by testing the effect of mutations In aim 2, I will demonstrate that ML297 binds at the interface between subunits in heteromeric channels and focus on the role of the GIRK2 subunit in ML297 activation of GIRK1/2 channels. Finally, in aim 3, I will examine the efficacy of ML297 at wild-type and mutant channels addressed in the first two aims using single channel recordings. Together, these experiments will isolate key regions of the GIRK channel important for activation by ML297 and establish a discrete mechanism for activating GIRK channels in the absence of GPCRs. Understanding the mechanism of allosteric activation of GIRK channels by anti-epileptic therapies could provide a novel pathway for the development of future anti-epileptic drugs, as well as potential therapies for other disorders such as addiction and Parkinson's disease, where GIRK channels have also been implicated as important drug targets.

描述(申请人提供)：癫痫影响了近300万美国人，10%的新患者未能通过正确的药物治疗控制癫痫发作。目前的抗癫痫疗法通过靶向大脑中的离子通道来恢复正常的神经传递。许多人对他们的处方产生耐受性，经历副作用，并需要随着时间的推移不断修改他们的治疗方案。今天，有一种内在的需求，即可以减少这些副作用并在更长时间内保持有效性的抗癫痫药物疗法。G蛋白内向整流钾(GIRK)通道调节大脑中的神经传递。由于它们在大脑中的区域分布，它们与许多神经疾病有关，包括成瘾、焦虑、癫痫和帕金森氏症。最近的研究表明，酒精可以通过N-末端、D-E和L-M形成的孤立的结合口袋直接激活GIRK通道，这个位于相邻亚基之间的结合部位可能是其他配体的靶标。ML297是一种抗癫痫的化合物，最近被发现并作为研究GIRK通道功能的工具发布。ML297激活GIRK通道的机制和通道上的结合位点都是未知的。分离这一机制可用于开发未来更好的抗癫痫治疗方法。我们假设ML297通过与酒精结合位点的直接结合而诱导通道激活。初步研究表明，ML297在缺乏GIRK1亚基的GIRK通道中不起作用。在这个方案中，我设计了一系列实验，在三个目标下，通过膜片钳电生理和定点突变分离ML297在不同GIRK通道上的结合位点。对于AIMS 1和2，我将使用全细胞膜片钳电生理学来检测ML297诱导的门控的宏观变化。在目标1中，我将通过测试目标2中突变的效果来建立ML297与GIRK1亚基结合的机制，我将证明ML297结合在异构体通道中的亚基之间的界面，并集中在GIRK2亚基在ML297激活GIRK1/2通道中的作用。最后，在目标3中，我将使用单通道录音来检验ML297在前两个目标中涉及的野生型和突变型通道的有效性。总之，这些实验将分离对ML297激活GIRK通道至关重要的关键区域，并建立一种在没有GPCRs的情况下激活GIRK通道的离散机制。了解抗癫痫药物对GIRK通道变构激活的机制可能为未来抗癫痫药物的开发提供新的途径，并为其他疾病的治疗提供潜在的治疗方法，如成瘾和帕金森病，在这些疾病中，GIRK通道也被认为是重要的药物靶点。