Structural and molecular basis of drug-induced IKACh reduction

描述（由申请人提供）：这份NIH支持申请旨在帮助我从目前的职业指导阶段过渡到独立。这将使我有机会学习结构和分子生物学的新概念和新技术，这将增加我在心脏电生理学方面的背景。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采用新颖的方法来改进现有的或产生新的药理疗法。我于2007年在纽约州立大学雪城分校药学系获得博士学位。我的论文集中在离子和体大小的心室颤动（VF）的起始和维持的决定因素。我阐明了肌上皮向内整流（Kir2.x）钾通道蛋白在VF维持中的作用，以及在His-浦肯野系统水平上ryanodine受体2型在室性心动过速起始中的作用。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，我一直在密歇根大学（U of M）心律失常研究中心做博士后。我还获得了美国心脏协会的博士后奖学金。在这里，我与密歇根大学的研究人员合作，从分子到器官，利用光学作图、贴片夹紧和分子模型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）和心室颤动（VF）的终止。我建议利用在密歇根大学随时可用的机会，将我在心脏电生理学方面的背景与我希望通过这项提案获得的新方法和技能结合起来，为自己开发一个科学利基。这个利基将不同于我过去的科学努力，但又与之互补，并将为我作为一名独立研究者的工作提供坚实的基础。我的建议源于这样一个前提，即抗心律失常药物-离子通道相互作用仍然知之甚少，不完整的知识和糟糕的药物设计可能是目前可用的抗心律失常药物无效的基础。形成负责乙酰胆碱激活钾电流（IKAch）通道的Kir3.1和Kir3.4蛋白对于维持AF基础上的转子非常重要。最近，Kir3.1细胞质结构域的晶体结构得到了解决，Kir3.1和Kir3.4运输的主要特征已经被描述。这提供了一个令人兴奋的机会，为通过IKACh减少导致AF终止的假定药物通道相互作用提供新的机制见解。我的假设是，IKACh的药理学减少可以通过两种机制实现：(1)直接通道阻断，涉及通道胞质区域的特定氨基酸；(2)通过Arf-6 GTPase依赖途径内化Kir3.1/Kir3.4异构体。我将利用氯喹作为模型药物，研究药物诱导的IKACh减少的结构和分子基础。氯喹是一种阻断IKACh的抗疟喹啉，已被证明可以终止一些患者的房颤。我的初步资料表明，氯喹：1-终止离体羊心脏胆碱能性心房颤动；2-通过与特定氨基酸残基的相互作用阻碍离子通过通道前庭的运动，如分子模型所示；核磁共振（NMR）实验表明，3-引起新生大鼠心房肌细胞Kir3.1/Kir3.4的内化，可能是通过与Kir3.4的羧基端酸性簇的直接相互作用。这些初步数据支持了我为验证我的假设而提出的实验的可行性。为了实现我的目标，我将采用多学科的方法，包括荧光显微镜、化学发光、核磁共振光谱、x射线晶体学和电生理学。这些综合研究代表了一个新的步骤，可以为合理设计心房特异性抗颤剂奠定基础。密歇根大学优越的环境是获得结构生物学和离子通道运输专业知识的理想场所。我将利用一流的设备和研究人员精通这些新领域。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明尼苏达大学结构生物学中心董事总经理Jeanne Stuckey博士的指导下获得必要的专业知识，1- x射线晶体学，我建议结晶并解决Kir3.1与氯奎复合物的高分辨率三维结构，2- Kir3.1/Kir3.4蛋白质运输的显微镜和生物化学。在明尼苏达大学药理学副教授Jeffery Martens博士和INSERM研究主任、法国巴黎皮埃尔·玛丽·居里大学Pitii-Salpitrihre医学院教授Stephane Hatem博士的指导下，研究了氯喹诱导的内化。通过结合我将学习的新技术和新概念，以及我将参加的晶体学和蛋白质学的相关课程和研讨会，我的导师将确保我向独立过渡。我将具备必要的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，即为改善抗纤颤设备做出贡献，和/或发现新的更有效的抗心律失常药物。

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.