Structural and molecular basis of drug-induced IKACh reduction

描述（由申请人提供）：此申请NIH支持旨在促进我从目前的职业生涯指导阶段向独立过渡。这将使我有机会学习结构和分子生物学方面的新概念和新技术，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的科学家，并研究药物离子通道相互作用的结构，功能和贩运方面。因此，我预见我的实验室将使用新颖的方法来改进现有的或产生新的药理学疗法。 我于2007年在纽约州立大学锡拉丘兹获得药理学博士学位。我的论文集中在离子和身体大小的决定因素室颤（VF）的启动和维持。我阐明了肌膜内向整流（Kir2.x）钾通道蛋白在维持VF中的作用，以及兰尼碱受体2型在希氏-浦肯野系统水平引发室性快速性心律失常中的作用。此外，我证明了转子是哺乳动物物种间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，从分子到器官，使用光学映射，膜片钳和分子建模来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电流的减少，并导致心房颤动（AF）和VF的终止。我建议利用在密歇根大学现成的机会，将我在心脏电生理学方面的背景与我希望通过这项提议获得的新方法和技能相结合，为自己开发一个科学利基。这个利基将不同于，但补充，我过去的科学努力，并将提供一个坚实的基础，我的工作作为一个独立的调查员。 我的建议源于这样一个前提，即抗糖尿病药物-离子通道相互作用仍然知之甚少，不完整的知识和糟糕的药物设计可能是目前可用的抗糖尿病药物无效的基础。Kir3.1和Kir3.4蛋白形成的通道负责乙酰胆碱激活的钾电流（IKAch）是重要的，在永存的转子，AF的基础。最近，Kir3.1胞质结构域的晶体结构得到解决，Kir3.1和Kir3.4贩运的主要特点已被描述。这提供了一个令人兴奋的机会，提供新的机制洞察推定的药物通道相互作用，导致房颤终止通过IKACh减少。我的假设是，IKACh的药理学降低可以通过两种机制实现：（1）直接通道阻断，涉及通道胞质结构域中的特定氨基酸;（2）通过Arf-6 GTP酶依赖性途径内化Kir3.1/Kir3.4异聚体。我将利用氯喹，一种抗疟喹啉阻断IKACh，并已被证明终止AF在一些患者中，作为模型药物研究药物诱导的IKACh减少的结构和分子基础。我的初步数据表明，氯喹：1-终止胆碱能AF在离体绵羊心脏2-阻碍离子运动通过通道的前庭通过与特定的氨基酸残基的分子模拟建议的相互作用; 3-导致新生大鼠心房肌细胞中Kir3.1/Kir3.4的内化，可能是通过与Kir3.4的羧基末端酸性簇的直接相互作用，如核磁共振（NMR）实验所示。这些初步的数据支持我提出的实验来检验我的假设的可行性。为了实现我的目标，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，X射线晶体学和电生理学。这些综合性研究代表了一个新的步骤，可以为心房特异性抗心律失常药物的合理设计奠定基础。 在M的U优秀的环境是理想的获得结构生物学和离子通道贩运的专业知识。我将利用恒星的设施和调查人员，精通这些新领域。我的导师Jose Jalife博士和其他导师制定的详细指导计划将确保我在密歇根大学结构生物学中心董事总经理Jeanne Stuckey博士的指导下获得1-X射线晶体学方面的必要专业知识，在那里我建议结晶和解决Kir3.1与氯喹复合的高分辨率3-D结构，和2-Kir3.1/Kir3.4蛋白质运输的显微镜和生物化学，以及它们在密歇根大学药理学副教授Jeffery Martens博士和巴黎皮埃尔玛丽居里大学Pitii-Salpitrihre医学院研究主任Stephane Hatem博士的指导下的氯喹诱导的内化，法国通过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的结合，我的导师将确保我向独立过渡。我将配备必要的资金和技能来创建一个实验室，专注于离子通道的结构/功能关系和贩运，这将有助于确保成功实现我的最终目标，即为改善抗癫痫药物和/或发现新的更有效的抗癫痫药物做出贡献。

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.