Structural and molecular basis of drug-induced IKACh reduction

摘要： 申请NIH支持的目的是促进我从目前的指导阶段 我的事业走向独立。这将使我有机会学习新的概念和技术 结构和分子生物学，我将在我的心脏电生理学背景中增加这些知识。我的长期任期 职业目标是成为一名独立的科学家，调查结构、功能和贩运 药物-离子通道相互作用方面。因此，我预见我的实验室将使用新的方法 致力于改进现有的或产生新的药理疗法。 我于2007年在纽约州立大学锡拉丘兹分校药理学系获得博士学位。我的论文 侧重于决定室颤(VF)发生和维持的离子和体型决定因素。我 阐明肌膜内向整流钾通道蛋白(Kir2.x)在维持心肌细胞功能中的作用 VF和兰尼定受体2型在组氨酸水平起始室性快速性心律失常中的作用。 浦肯野系统。此外，我还证明了转子是哺乳动物发生室颤的机制。 自2008年以来，我一直是密歇根大学心律失常中心的博士后研究员 研究。我还获得了美国心脏协会博士后奖学金。在这里我与密歇根大学合作 M调查人员致力于从分子到器官阐明氯喹和氯喹之间的相互作用 使用光学映射、膜片钳和分子建模的内向整流通道。这样的互动 导致内向整流电流减少，并导致房颤和室颤的终止。我 我建议利用密歇根大学现成的机会，结合我在心脏领域的背景 电生理学，我希望通过这个提议获得新的方法和技能，以开发一种 对我自己来说是个科学利基。这个利基市场将与我过去的科学研究不同，但却是对我的赞扬 努力，并将为我作为一名独立调查员的工作提供坚实的基础。 我的建议源于这样一个前提，即抗心律失常药物-离子通道的相互作用仍然很差 了解，不完全的知识和糟糕的药物设计可能是目前药物无效的原因 可用的抗心律失常药物。Kir3.1和Kir3.4蛋白形成了负责 乙酰胆碱激活的钾电流(IKAch)在房颤下的转子永久化过程中起着重要作用。 最近，Kir3.1胞质结构域的晶体结构被解决，Kir3.1和Kir3.1的主要特征 Kir3.4描述了贩卖人口的情况。这提供了一个令人兴奋的机会来提供新的机械洞察力 转化为可能的药物-通道相互作用，从而通过IKACh还原导致房颤终止。我的假设是 IKACh的药理作用可通过两种机制实现：(1)直接阻断通道 在通道的细胞质区域涉及特定的氨基酸；以及(2)Kir3.1/Kir3.4的内化 通过Arf-6 GTPase依赖途径的异构体。我会用氯喹，一种抗疟疾的喹啉 这阻断了IKACh，并已被证明在一些患者中终止房颤，作为研究 药物诱导IKACh还原的结构和分子基础。我的初步数据显示，氯喹：1- 终止离体羊心脏胆碱能房颤；2-阻碍离子通过通道的运动 前庭通过与特定氨基酸残基的相互作用，如分子模型所建议的；3-导致 Kir3.1/Kir3.4在乳鼠心房肌细胞中的内化，可能是通过与Kir3.1/Kir3.4的直接相互作用 核磁共振实验表明，Kir3.4的羧基末端为酸性簇。 这些初步数据支持了我提出的用来检验我的假设的实验的可行性。要实现 我的目标，我将使用多学科的方法，涉及荧光显微镜，化学发光，核磁共振 光谱学、X射线结晶学和电生理学。这些综合研究代表了一个新的步骤 这为合理设计心房特异性抗纤颤药物奠定了基础。 密歇根大学优越的环境是获得结构生物学和离子专业知识的理想选择 渠道贩运。我将利用星级设施和调查人员精通这些新的 菲尔兹。我的导师Jose Jalife博士和共同导师制定的详细指导计划将确保我将 在珍妮·斯塔基博士的指导下获得必要的1-X-射线结晶学专业知识， 密歇根大学结构生物学中心的常务董事，我建议在那里结晶并解决一个高 Kir3.1与氯喹配合物的三维分辨结构及其二维显微镜和生化研究 Kir3.1/Kir3.4蛋白的运输及其在Dr. 密歇根大学药理学副教授杰弗里·马滕斯和斯蒂芬·哈特姆博士 INSERM研究，Pierre Marie Piti-Salp Trire医学院教授 法国巴黎的居里大学。通过我将学到的新技术和新概念的结合， 我将参加有关结晶学和蛋白质组学的课程和研讨会，我的导师将 确保我过渡到独立。我将配备必要的资金和技能来创建一个实验室 重点研究离子通道的结构/功能关系和运输，这将有助于确保成功 实现我的最终目标，即为改进抗纤颤医疗设备做出贡献，和/或 更有效的抗心律失常新药的发现。

SUMMARY: This application for NIH support is aimed at facilitating my transition from the current mentored stage of my career toward independence. It will give me the opportunity to learn new concepts and techniques in structural and molecular biology, which I will add to my background in cardiac electrophysiology. My long term career objective is to be an independent scientist, and to investigate structural, functional and trafficking aspects of drug-ion channels interactions. Therefore, I foresee that my laboratory will use novel approaches geared towards improving existing or generating new pharmacological therapies. I obtained my PhD from the Department of Pharmacology at SUNY Syracuse in 2007. My thesis focused on ionic and body size determinants of ventricular fibrillation (VF) initiation and maintenance. I elucidated the roles of sarcolemmal inward rectifier (Kir2.x) potassium channel proteins in the maintenance of VF, and of the ryanodine receptor type 2 in the initiation of ventricular tachyarrhythmias at the level of the His- Purkinje system. Additionally, I demonstrated that rotors are the mechanism of VF across mammalian species. Since 2008, I have been a postdoctoral fellow at the University of Michigan (U of M) Center for Arrhythmia Research. I also received an American Heart Association Postdoctoral Fellowship. Here I collaborate with U of M investigators towards elucidating, from the molecule to the organ, the interactions between chloroquine and inward rectifier channels using optical mapping, patch clamping and molecular modeling. Such interactions result in the reduction of inward rectifier currents, and lead to the termination of atrial fibrillation (AF) and VF. I propose to take advantage of opportunities readily available at U of M to combine my background in cardiac electrophysiology with new methodologies and skills that I hope to acquire through this proposal, to develop a scientific niche for myself. That niche will be dissimilar from, yet complimentary to, my past scientific endeavors, and will provide a solid basis of my work as an independent investigator. My proposal stems from the premise that antiarrhythmic drug-ion channel interactions remain poorly understood, and that incomplete knowledge and poor drug design may underlie the inefficacy of currently available antiarrhythmics. The Kir3.1 and Kir3.4 proteins that form the channels responsible for the acetylcholine-activated potassium current (IKAch) are important in perpetuating the rotors that underlie AF. Recently, the crystal structure of the Kir3.1 cytoplasmic domain was solved and the main features of Kir3.1 and Kir3.4 trafficking have been described. This offers an exciting opportunity to provide novel mechanistic insight into putative drug-channel interactions that result in AF termination through IKACh reduction. My hypothesis is that pharmacological reduction of IKACh can be achieved through two mechanisms: (1) direct channel blockade involving specific amino acids in the cytoplasmic domain of the channel; and (2) internalization of Kir3.1/Kir3.4 heteromers through the Arf-6 GTPase dependent pathway. I will utilize chloroquine, an antimalarial quinoline that blocks IKACh, and has been shown to terminate AF in some patients, as a model agent to study the structural and molecular basis of drug-induced IKACh reduction. My preliminary data indicate that chloroquine: 1- terminates cholinergic AF in the isolated sheep heart; 2- impedes ion movement through the channel's vestibule by interacting with specific amino acid residues as suggested by molecular modeling; 3- causes the internalization of Kir3.1/Kir3.4 in neonatal rat atrial myocytes, possibly through a direct interaction with the carboxyl terminus acidic cluster of Kir3.4, as suggested by nuclear magnetic resonance (NMR) experiments. These preliminary data support the feasibility of the experiments I propose to test my hypothesis. To achieve my aims, I will use a multidisciplinary approach, involving fluorescence microscopy, chemiluminescence, NMR spectroscopy, X-ray crystallography and electrophysiology. These integrative studies represent a novel step that can set the stage for the rational design of atrial-specific antifibrillatory agents. The outstanding environment at the U of M is ideal for attaining expertise in structural biology and ion channel trafficking. I will make use of the stellar facilities and investigators to become proficient in these new fields. The detailed mentoring plan laid out by my mentor, Dr. Jose Jalife, and co-mentors will ensure that I will acquire the necessary expertise in 1- X-ray crystallography under the guidance of Dr. Jeanne Stuckey, managing director of the Center for Structural biology at U of M, where I propose to crystallize and solve a high resolution 3-D structure of Kir3.1 in complex with chloroquine, and 2- microscopy and biochemistry of trafficking of Kir3.1/Kir3.4 proteins, and their chloroquine-induced internalization under the mentorship of Dr. Jeffery Martens, Associate Professor of Pharmacology at U of M, and Dr. Stephane Hatem, Director of Research at the INSERM, and Professor at the Faculty of Medicine Piti¿-Salp¿tri¿re of the Pierre Marie Curie University in Paris, France. Through the combination of the new techniques and concepts I will learn, and the relevant courses and seminars in crystallography and proteonomics I will attend, my mentors will ensure my transition to independence. I will be equipped with the wherewithal and skill to create a laboratory focused on structure/function relations and trafficking of ion channels, which will help to ensure the successful attainment of my ultimate goal of contributing to the improvement of the antifibrillatory armamentarium, and/or the discovery of new more effective antiarrhythmic drugs.