Real Time Screening for GIRK1/4 Channel Blockers

描述（由申请人提供）：心房颤动（AF）是最常见的心律失常，是造成重大发病率、死亡率和医疗费用的原因。随着人口的老龄化，房颤的发病率预计将显著增加。然而，传统的抗心律失常药物治疗AF由于其促心律失常的作用而受到限制。房颤药物治疗的一个新靶点是乙酰胆碱激活的K+通道（IK,Ach）。该通道是被称为G蛋白偶联内向整流K+ (GIRK)通道的蛋白质超家族的成员，由GIRK1/4亚基组成。乙酰胆碱与心肌毒蕈碱（M2）受体结合后，乙酰胆碱打开，并介导迷走神经对热率和心房兴奋的影响。最近的研究表明，IK，Ach在房颤中具有组成性活性，从而增强心房兴奋性。该项目的目标是开发一种高通量筛选（HTS）方法，用于鉴定抑制IK，Ach的新药。本项目要测试的假设是，膜电位敏感染料可以作为HTS传感器用于筛选永活心肌细胞系中的IK，Ach阻滞剂。在Aim #1中，将建立使用荧光成像板读取器（FLIPR）测量和分析IK，Ach的方法。在Aim #2中，将开发高密度HTS检测，并使用离子通道阻滞剂库进行筛选。在实验中发现的新化合物将在分离的AF大鼠心脏模型中进行测试。总的来说，实验室工作的长期目标是开发治疗心律失常的新治疗策略。

DESCRIPTION (provided by applicant): Atrial fibrillation (AF) is the most commonly occurring cardiac arrhythmia and is responsible for significant morbidity, mortality and health care costs. The incidence of AF is expected to increase markedly with the aging population. However, the use of conventional anti-arrhythmic drugs for treating AF has been limited due to their pro- arrhythmic actions. One novel target for AF drug therapy is the acetylcholine-activated K+ channel (IK,Ach). This channel is a member of the super-family of proteins known as the G protein-coupled inward rectifier K+ (GIRK) channels and is composed of the GIRK1/4 subunits. IK,Ach opens upon binding of acetylcholine to the cardiac muscarinic (M2) receptor and mediates vagal effects on heat rate and atrial excitation. Recent studies indicate that IK,Ach is constitutively active in AF, thus enhancing atrial excitability. The goal of this project is to develop a high throughput-screening (HTS) assay for identifying new drugs that inhibit IK,Ach. The hypothesis to be tested in this project is that membrane potential-sensitive dyes can be used as a HTS sensor in screening for IK,Ach blockers in immortalized cardiac cell lines. In Aim #1 methods for the measurement and analysis of IK,Ach, using a fluorescent imaging plate reader (FLIPR), will be established. In Aim #2 a high density HTS assay will be developed and screened with a library of ion channel blockers. New compounds identified in the assay will be tested in an isolated rat heart model of AF. Overall, the long term goal of work in the laboratory is to develop novel therapeutic strategies for treating cardiac arrhythmias. PUBLIC HEALTH RELEVANCE: Atrial fibrillation (AF) is the most commonly occurring cardiac arrhythmia and is responsible for significant morbidity, mortality and health care costs. The incidence of AF is expected to increase markedly with the aging population. The goal of this project is to identify new drugs for treating AF.