Structural and molecular basis of drug-induced IKACh reduction

药物诱导的 IKACh 减少的结构和分子基础

• 批准号: 8535190
• 负责人: Sami Fouad Noujaim
• 金额: $ 23.21万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8535190
• 关键词: 3-Dimensional; Acetylcholine; Action Potentials; Affect; American Heart Association; Amino Acids; Anti-Arrhythmia Agents; Antimalarials; Arrhythmia; Atrial Fibrillation; Binding; Biochemistry; Body Size; Cardiac; Cell membrane; Cells; Charge; Chemiluminescence assay; Chloroquine; Complex; Crystallization; Crystallography; Cytoplasmic Tail; Data; Docking; Doctor of Philosophy; Dose; Drug Design; Electrophysiology (science); Endoplasmic Reticulum; Ensure; Environment; Faculty; Failure; Fellowship; Fluorescence Microscopy; Frequencies; Goals; Golgi Apparatus; Grant; Guanosine Triphosphate Phosphohydrolases; Heart; Heart Atrium; Immunofluorescence Immunologic; Ion Channel; Ions; Knowledge; Laboratories; Lead; Learning; Maintenance; Maps; Martens; Mediating; Medicine; Mentors; Mentorship; Methodology; Michigan; Microscopy; Modeling; Molecular; Molecular Biology; Molecular Models; Morbidity - disease rate; Movement; Muscle Cells; Mutagenesis; Mutate; NMR Spectroscopy; Neonatal; Nuclear Magnetic Resonance; Optics; Organ; Paris, France; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacology; Postdoctoral Fellow; Potassium; Potassium Channel; Proteins; Rattus; Relative (related person); Research; Research Personnel; Resolution; Role; Ryanodine Receptor Calcium Release Channel; Scientist; Sheep; Side; Signal Transduction; Solid; Staging; Structure; Surface; System; Tachyarrhythmias; Techniques; Testing; United States National Institutes of Health; Universities; Ventricular; Ventricular Fibrillation; Vestibule; Work; X-Ray Crystallography; base; career; cholinergic; design; improved; insight; interdisciplinary approach; molecular modeling; monolayer; mortality; mutant; novel; novel strategies; overexpression; patch clamp; professor; prototype; quinoline; receptor; research study; response; simulation; skills; stem; structural biology; trafficking

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.

概括： 此 NIH 支持申请旨在促进我从当前的指导阶段过渡 我的职业走向独立将使我有机会学习新的概念和技术。 结构和分子生物学，我将把它添加到我的长期心脏电生理学背景中。 职业目标是成为一名独立科学家，并研究结构、功能和贩运 因此，我预见我的实验室将使用新的方法。 旨在改善现有或产生新的药物疗法。 我于 2007 年在纽约州立大学雪城分校药理学系获得博士学位。我的论文 重点关注心室颤动 (VF) 发生和维持的离子和体型决定因素 I。 阐明了肌膜内向整流器（Kir2.x）钾通道蛋白在维持 VF 和 2 型兰尼碱受体在组氨酸水平引发室性快速心律失常中的作用 此外，我还证明了转子是哺乳动物物种 VF 的机制。 自2008年起，我一直在密歇根大学（U of M）心律失常中心担任博士后研究员 我还获得了美国心脏协会的博士后奖学金。 M 研究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、膜片钳和分子建模等相互作用来构建内向整流器通道。 导致内向整流电流减少，并导致心房颤动（AF）和 VF I 的终止。 建议利用密歇根大学现有的机会结合我在心脏病方面的背景 我希望通过本提案获得电生理学的新方法和技能，以开发 我自己的科学定位与我过去的科学定位不同，但却是互补的。 努力，并将为我作为独立调查员的工作奠定坚实的基础。 我的建议源于抗心律失常药物离子通道相互作用仍然很差的前提 理解，不完整的知识和不良的药物设计可能是目前无效的基础 可用的抗心律失常药物 Kir3.1 和 Kir3.4 蛋白形成负责的通道。 乙酰胆碱激活钾电流 (IKAch) 对于维持 AF 下的转子的存在非常重要。 最近，Kir3.1胞质结构域的晶体结构被解析，Kir3.1和Kir3.1的主要特征 Kir3.4 贩运已被描述，这提供了一个令人兴奋的机会来提供新颖的机制见解。 推定的药物通道相互作用，通过 IKACh 减少导致 AF 终止。 IKACh 的药理减少可以通过两种机制实现：（1）直接通道阻断 (2) Kir3.1/Kir3.4 的内化 我将利用氯喹，一种抗疟喹啉药物。 阻断 IKACh，并已被证明可以终止某些患者的 AF，作为研究 药物诱导的 IKACh 减少的结构和分子基础 我的初步数据表明氯喹：1- 终止离体绵羊心脏中的胆碱能 AF；2- 阻碍离子通过通道的运动； 如分子模型所示，通过与特定氨基酸残基相互作用，导致前庭； Kir3.1/Kir3.4 在新生大鼠心房肌细胞中的内化，可能是通过与 核磁共振 (NMR) 实验表明，Kir3.4 的羧基末端酸性簇。 这些初步数据支持了我提出的实验的可行性，以检验我的假设。 我的目标是采用多学科方法，包括荧光显微镜、化学发光、核磁共振 光谱学、X 射线晶体学和电生理学这些综合研究代表了一个新的进步。 这可以为心房特异性抗纤颤药物的合理设计奠定基础。 密歇根大学优越的环境是获得结构生物学和离子学专业知识的理想选择 我将利用一流的设施和调查员来精通这些新的内容。 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能够做到这一点。 在 Jeanne Stuckey 博士的指导下获得 1-X 射线晶体学方面必要的专业知识， 密歇根大学结构生物学中心常务董事，我建议在那里结晶并解决一个高 Kir3.1 与氯喹复合物的分辨率 3-D 结构，以及 2- 显微镜和生物化学 在 Dr. 的指导下，Kir3.1/Kir3.4 蛋白的贩运及其氯喹诱导的内化 Jeffery Martens，密歇根大学药理学副教授，Stephane Hatem 博士，主任 INSERM 的研究和 Piti 医学院的教授-樽海鞘??三??皮埃尔玛丽的re 法国巴黎居里大学通过结合我将学习的新技术和概念， 以及我将参加的晶体学和蛋白质组学的相关课程和研讨会，我的导师将 确保我过渡到独立。我将具备创建实验室的资金和技能。 重点关注离子通道的结构/功能关系和运输，这将有助于确保成功 实现我的最终目标，即为改进抗纤颤武器做出贡献，和/或 发现新的更有效的抗心律失常药物。