Structural and molecular basis of drug-induced IKACh reduction

总结： 这个申请NIH支持的目的是为了促进我从目前的指导阶段过渡， 我的事业走向独立这将使我有机会学习新的概念和技术， 结构和分子生物学，我将把它加入我的心脏电生理学背景。我的长期 职业目标是成为一名独立的科学家，并调查结构，功能和贩运 药物-离子通道相互作用方面。因此，我预见我的实验室将使用新的方法， 旨在改善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，和ryanodine受体2型在启动室性快速性心律失常在水平的His- 浦肯野系统。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）心律失常中心担任博士后研究员 Research.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， M研究人员致力于阐明，从分子到器官，氯喹和 内向整流器通道使用光学映射，膜片钳和分子建模。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 我建议利用在密歇根大学的机会，结合我在心脏方面的联合收割机背景 电生理学与新的方法和技能，我希望通过这项建议获得，发展一个 我自己的科学利基。这个利基将不同于，但补充，我过去的科学 努力，并将为我作为独立调查员的工作提供坚实的基础。 我的建议源于这样一个前提，即抗肿瘤药物-离子通道相互作用仍然很差， 不完整的知识和糟糕的药物设计可能是目前药物无效的原因。 可用的抗氧化剂。Kir3.1和Kir3.4蛋白形成了负责 乙酰胆碱激活的钾电流（IKAch）是重要的永久转子AF的基础。 最近，Kir3.1胞质结构域的晶体结构被解决，Kir3.1和Kir3.2的主要特征被发现。 Kir3.4贩运已被描述。这提供了一个令人兴奋的机会， 通过IKACh减少导致AF终止的假定药物通道相互作用。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 涉及通道的胞质结构域中的特定氨基酸;和（2）Kir3.1/Kir3.4的内化 通过Arf-6 GTP酶依赖性途径，我会使用氯喹一种抗疟疾的喹啉 阻断IKACh，并已被证明在一些患者中终止AF，作为研究 药物诱导IKACh减少的结构和分子基础。我的初步数据显示氯喹：1- 终止离体羊心脏中的胆碱能AF; 2-阻碍离子通过通道的运动 前庭通过与特定的氨基酸残基相互作用，如分子模拟所示; 3-引起前庭 Kir3.1/Kir3.4在新生大鼠心房肌细胞中的内化，可能是通过与 Kir3.4的羧基末端酸性簇，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。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步骤 这可以为心房特异性抗炎症剂的合理设计奠定基础。 杰出的环境在密歇根大学是理想的获得专业知识的结构生物学和离子 渠道贩运。我将利用恒星的设施和调查人员，成为精通这些新的 领域的我的导师Jose Jalife博士和其他导师制定的详细指导计划将确保我 在Jeanne Stuckey博士的指导下获得1-X射线晶体学的必要专业知识， 我是密歇根大学结构生物学中心的总经理，我建议在那里结晶和解决一个高层次的问题。 Kir3.1与氯喹复合物的三维结构，以及Kir3.1与氯喹复合物的二维显微镜和生物化学。 Kir3.1/Kir3.4蛋白的运输，以及在Dr. 密歇根大学药理学副教授Jeffery Martens和 研究在PIMM，并在医学系教授皮蒂-萨尔普re的皮埃尔玛丽 法国巴黎的居里大学。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 确保我的独立过渡我将拥有必要的资金和技术来建立一个实验室 着重于结构/功能关系和离子通道的运输，这将有助于确保成功的 实现我的最终目标，为改善防辐射设备做出贡献，和/或 新的更有效的抗疟疾药物的发现。

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.