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: 8028282
• 负责人: Sami Fouad Noujaim
• 金额: $ 9.65万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 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 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8028282
• 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; Health; 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

DESCRIPTION (provided by applicant): 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 Pitii-Salpitrihre 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.

描述（由申请人提供）：这份 NIH 支持申请旨在促进我从当前职业生涯的指导阶段过渡到独立。它将让我有机会学习结构和分子生物学的新概念和技术，我将把它们添加到我的心脏电生理学背景中。我的长期职业目标是成为一名独立科学家，研究药物离子通道相互作用的结构、功能和运输方面。因此，我预见我的实验室将使用旨在改进现有或产生新的药物疗法的新方法。 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。我的论文重点是心室颤动 (VF) 发生和维持的离子和体型决定因素。我阐明了肌膜内向整流 (Kir2.x) 钾通道蛋白在维持室颤中的作用，以及 2 型兰尼碱受体在希斯-浦肯野系统水平引发室性快速性心律失常中的作用。此外，我还证明了转子是哺乳动物物种 VF 的机制。自2008年以来，我一直在密歇根大学（U of M）心律失常研究中心担任博士后研究员。我还获得了美国心脏协会博士后奖学金。在这里，我与密歇根大学的研究人员合作，利用光学测绘、膜片钳和分子建模从分子到器官阐明氯喹和内向整流通道之间的相互作用。这种相互作用导致内向整流器电流减少，并导致心房颤动 (AF) 和 VF 的终止。我建议利用密歇根大学现有的机会，将我在心脏电生理学方面的背景与我希望通过此提案获得的新方法和技能相结合，为自己开发一个科学领域。这个利基市场将与我过去的科学事业有所不同，但却是互补的，并将为我作为一名独立研究者的工作提供坚实的基础。 我的提议源于这样一个前提：抗心律失常药物-离子通道相互作用仍然知之甚少，不完整的知识和不良的药物设计可能是目前可用的抗心律失常药物无效的原因。 Kir3.1 和 Kir3.4 蛋白形成负责乙酰胆碱激活钾电流 (IKAch) 的通道，对于维持 AF 下的转子至关重要。最近，Kir3.1胞质结构域的晶体结构被解析，并且描述了Kir3.1和Kir3.4运输的主要特征。这提供了一个令人兴奋的机会，可以为推定的药物通道相互作用提供新的机制见解，通过 IKACh 减少导致 AF 终止。我的假设是，IKACh 的药理减少可以通过两种机制来实现：（1）直接通道阻断，涉及通道胞质域中的特定氨基酸； (2) Kir3.1/Kir3.4 异聚体通过 Arf-6 GTPase 依赖性途径内化。我将利用氯喹（一种阻断 IKACh 的抗疟喹啉，已被证明可以终止某些患者的 AF）作为模型药物来研究药物诱导的 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- 如分子模型所示，通过与特定氨基酸残基相互作用，阻碍离子通过通道前庭的运动； 3-如核磁共振 (NMR) 实验所示，可能通过与 Kir3.4 的羧基末端酸性簇直接相互作用，导致新生大鼠心房肌细胞中 Kir3.1/Kir3.4 的内化。这些初步数据支持了我提议检验我的假设的实验的可行性。为了实现我的目标，我将采用多学科方法，包括荧光显微镜、化学发光、核磁共振波谱、X 射线晶体学和电生理学。这些综合研究代表了一个新颖的步骤，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非常适合获得结构生物学和离子通道运输方面的专业知识。我将利用一流的设施和研究人员来精通这些新领域。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在中心总经理 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。密歇根大学的结构生物学，我建议结晶并解析 Kir3.1 与氯喹复合物的高分辨率 3-D 结构，以及 2- 显微镜和生物化学的运输Kir3.1/Kir3.4 蛋白及其氯喹诱导的内化，在密歇根大学药理学副教授 Jeffery Martens 博士和 INSERM 研究主任兼法国巴黎皮埃尔·玛丽·居里大学医学院 Pitii-Salpitrihre。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，我的导师将确保我向独立的过渡。我将具备资金和技能来创建一个专注于结构/功能关系和离子通道运输的实验室，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。