Molecular biophysics of cAMP regulation in HCN channels

HCN 通道中 cAMP 调节的分子生物物理学

基本信息

批准号：
9212819
负责人：
Qinglian Liu
金额：
$ 30.55万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9212819
关键词：
Address Affect Affinity Allosteric Regulation Anxiety Disorders Arrhythmia Behavior Belief Binding Binding Proteins Biochemical Biochemistry Biological Assay Biophotonics Biophysics Brain C-terminal Cardiovascular system Cereals Chemicals Computational Biology Computing Methodologies Coupling Critical Pathways Crystallization Cues Cyclic AMP Cyclic Nucleotides Distal Electrophysiology (science)Elements Enzymes Epilepsy Equilibrium Esthesia Fluorometry Foundations Frequencies Goals HCN4 gene Heart Heart Diseases Human Ion Channel Ion Channel Protein Ions Length Ligand Binding Ligands Link Measures Membrane Membrane Potentials Methods Molecular Molecular Conformation Mood Disorders Motion Mutation Neurons One-Step dentin bonding system Pacemakers Pain Physiological Play Protein Conformation Protein Dynamics Protein Isoforms Proteins Regulation Research Resolution Rest Role Short-Term Memory Signal Pathway Skeleton Solid Stimulus Structure Surface Surveys Targeted Research Techniques Time Touch sensation Travel Water Work analog biophysical analysis cardiac pacing design disease-causing mutation human disease insight macromolecule millisecond mutant nervous system disorder patch clamp protein folding protein function protein structure public health relevance response single molecule structural biology vibration voltage

项目摘要

DESCRIPTION (provided by applicant): HCN channels play important physiological functions in the brain and heart, from working memory formation, pain sensation, to cardiac pace making. HCN channels sense both electrical and chemical stimuli and bridges membrane excitability with intracellular signaling pathways. Dually regulated by voltage and ligand binding, HCN channel forms an elegant research target for protein allostery. Intracellular cAMP directly binds to and opens the channel. The basic question of how cAMP binding opens the channel remains elusive. We approach this research topic by following the research theme of structure, dynamics, and function. We have made significant progress by solving the crystal structures for the WT and a mutant form of human HCN4 C- terminal fragment, which contains the cyclic nucleotide binding domain (CNBD). To address the dynamic interaction between cAMP and the whole channel, we established the patch-clamp fluorometry technique that provides simultaneous recordings of channel activity and cAMP. We demonstrated that cAMP preferably binds to the channel in the open state. Then we went one step further and investigated how distributed sub-domains contribute to the global binding of cAMP. We found that the inner activation gate in the ion conducting pore remotely controls cAMP binding. This exciting discovery directly touches upon the very basics of how ligand- dependent regulation of protein functions is implemented. A fundamental understanding of the protein allostery in cAMP regulation of HCN channel is still missing. We are propelled to expand our study by the following two challenges. First, given the detailed understanding of isolated domains within the protein molecule, how they communicate with each other and the rest of the protein remains largely unknown. Secondly, for the study of protein allostery, it s challenging but critical to integrate the information from both structure and dynamics. To circumvent these difficulties, we have established and applied the techniques of electrophysiology, biophotonics, biochemistry, structural and computational biology. We have three specific aims: 1) Interpret allosteric ligand regulation at the level of liand - whole protein interaction. We will study the dynamic, cAMP - whole channel interaction in other HCN isoforms and the roles of important sub-domains, including the S4-S5 linker and C-linker, in remotely affecting cAMP binding. 2) Solve structures representing transitional states in cAMP gating. We will pursue the structure of the unliganded form and mutant forms of the protein and continue our effort in pursuing the full-length HCN structures. 3) Investigate the intriguing relationship between protein structure and dynamics. To this end, we will combine computational and experimental approaches for protein dynamics to address the molecular motions that define the direction of conformation changes during cAMP regulation of HCN channel. This proposal will lay a strong foundation for our long-ter research goals: 1) a fundamental understanding of protein allostery and protein folding, using ion channel proteins as a research platform~ 2) insights for the treatment of ion channel related neurological and cardiac disorders.

描述（由申请人提供）：HCN通道在大脑和心脏中起着重要的生理功能，从工作记忆形成，疼痛感到心脏速度。 HCN通道感知电和化学刺激，并通过细胞内信号通路桥接膜兴奋性。 HCN通道受电压和配体结合的双重调节，构成了蛋白质变构的优雅研究目标。细胞内营地直接与通道结合并打开。营地如何打开渠道的基本问题仍然难以捉摸。我们通过遵循结构，动力学和功能的研究主题来处理此研究主题。我们通过解决了含有循环核苷酸结合结构域（CNBD）的人类HCN4 c-末端片段的WT和突变形式的晶体结构，从而取得了重大进展。为了解决CAMP与整个通道之间的动态相互作用，我们建立了贴片钳荧光灯计量学技术，该技术提供了同时记录通道活动和CAMP的记录。我们证明了营地最好在开放状态下与通道结合。然后，我们又走了一步，并调查了分布式子域是如何促进CAMP的全球结合的。我们发现，导电孔中的内部激活门会远程控制cAMP结合。这一令人兴奋的发现直接涉及如何实施配体依赖性调节的基础知识。对HCN通道cAMP调节中蛋白质变构的基本理解仍然缺失。我们被推动通过以下两个挑战扩大研究。首先，鉴于对蛋白质分子中孤立域的详细理解，它们如何相互交流，其余蛋白质仍然在很大程度上未知。其次，对于蛋白质变构的研究，整合来自结构和动力学的信息的挑战但至关重要。为了避免这些困难，我们已经建立并应用了电生理学，生物探针，生物化学，结构和计算生物学的技术。我们有三个特定的目的：1）在LIAND - 全蛋白质相互作用水平上解释变构配体调节。我们将研究其他HCN同工型中的动态，cAMP-整个通道相互作用以及重要的子域的作用，包括S4-S5接头和C-Linker，在远程影响CAMP结合中。 2）求解代表营地过渡状态的结构。我们将追求蛋白质的非物体形式和突变形式的结构，并继续努力追求全长的HCN结构。 3）研究蛋白质结构与动力学之间的有趣关系。为此，我们将结合蛋白质动力学的计算和实验方法，以解决定义HCN通道cAMP调节过程中构象变化方向的分子运动。该建议将为我们的长期研究目标奠定坚实的基础：1）对蛋白质变构和蛋白质折叠的基本理解，使用离子通道蛋白作为研究平台〜2）用于治疗离子的见解通道相关的神经和心脏疾病。