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: 8208063
• 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 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8208063
• 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： NIH支持的申请旨在促进我从当前重要阶段的过渡 我朝着独立的职业。这将使我有机会学习新的概念和技术 结构和分子生物学，我将添加到心脏电生理学的背景中。我的长期 职业目标是成为一名独立科学家，并调查结构，功能和贩运 毒品离子通道相互作用的各个方面。因此，我预见我的实验室将使用新颖的方法 旨在改善现有或生成新的药物疗法。 我于2007年从锡拉丘兹（Suny Syracuse）的药理学系获得了博士学位。 专注于离子和体型确定心室纤颤（VF）倡议和维护。我 阐明了肌膜内部整流器（KIR2.X）钾通道蛋白在维持中的作用 VF，以及2型ryanodine受体在室中室里的心律不齐的启动时 Purkinje系统。此外，我证明了转子是跨哺乳动物物种的VF机制。 自2008年以来，我一直是密歇根大学（U of M）心律不齐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U合作 研究人员阐明从分子到器官，氯喹与器官之间的相互作用 使用光学映射，贴片夹具和分子建模的内向整流器通道。这样的互动 导致内向整流器电流的减少，并导致房颤（AF）和VF的终止。我 提议利用M的U随时获得的机会，以将我的背景结合在心脏中 我希望通过该提案获得的新方法和技能的电生理学，以开发一个 我自己的科学利基。那个利基市场与我过去的科学不相同，但又互补 努力，并将为我作为独立研究者的工作提供扎实的基础。 我的提议步骤从抗心律失常药物通道相互作用保持较差的前提下 理解，不完整的知识和药物设计可能是当前的效率低下的基础 可用的抗心律失常。构成负责渠道的Kir3.1和Kir3.4蛋白 乙酰胆碱激活的钾电流（IKACH）对于使AF构成的转子永久存在很重要。 最近，解决了Kir3.1细胞质结构域的晶体结构，并将Kir3.1和 已经描述了Kir3.4贩运。这提供了一个令人兴奋的机会来提供新颖的机械洞察力 进入推定的药物通道相互作用，从而导致通过减少IKACH终止AF。我的假设是 可以通过两种机制来实现IKACH的药物减少：（1）直接通道封锁 涉及通道的细胞质结构域中的特定氨基酸； （2）Kir3.1/Kir3.4的内在化 通过ARF-6 GTPase依赖性途径进行异域。我将利用氯喹，一种抗疟疾喹啉 这阻碍了ikach，并已被证明在某些患者中终止AF，作为研究的模型剂 药物诱导的IKACH还原的结构和分子基础。我的初步数据表明氯喹：1- 在孤立的绵羊心中终止胆碱能AF； 2条阻滞离子运动穿过频道的运动 通过与特定氨基酸相互作用，如分子建模所建议的那样保留； 3-导致 Kir3.1/Kir3.4在新生大鼠心房肌细胞中的内在化，可以通过与 如核磁共振（NMR）实验所示，KIR3.4的羧基酸性簇。 这些初步数据支持我提出的测试假设的实验的可行性。实现 我的目的是，我将使用涉及荧光显微镜，化学发光，NMR的多学科方法 光谱，X射线晶体学和电生理学。这些综合研究代表了一个新的步骤 这可以为房屋特异性防颤振剂的合理设计奠定基础。 M的U的杰出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。我将利用出色的设施和调查人员精通这些新的 字段。我的心理，何塞·贾利弗（Jose Jalife）博士和联合委员会制定的详细心理计划将确保我将 在Jeanne Stuckey博士的指导下获得1-X射线晶体学的必要专业知识， U的U的结构生物学中心董事总经理，我建议在这里结晶和解决高度 kir3.1分辨率3-D结构与氯喹，2-显微镜和生物化学 在Dr. U的U级药理学副教授Jeffery Martens和Stephane Hatem博士 Inserm的研究，Pierre Marie的医学学院教授 法国巴黎的居里大学。通过结合新技术和概念，我将学习， 以及我将参加的晶体学和蛋白质学方面的相关课程和半段，我的导师将 确保我向独立过渡。我将等同于创建实验室的技巧和技能 专注于结构/功能关系和离子渠道的贩运，这将有助于确保成功 实现我的最终目标是为改善防颤振武器库的改善和/或 发现新的更有效的抗心律失常药物。

项目成果

Polyunsaturated Fatty acids in atrial fibrillation: looking for the proper candidates.

心房颤动中的多不饱和脂肪酸：寻找合适的候选者。

• DOI: 10.3389/fphys.2012.00370
• 发表时间: 2012
• 期刊: Frontiers in physiology
• 影响因子: 4
• 作者: Salvador-Montañés,Oscar;Gómez-Gallanti,Alfonso;Garofalo,Daniel;Noujaim,SamiF;Peinado,Rafael;Filgueiras-Rama,David
• 通讯作者: Filgueiras-Rama,David