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发挥着重要的生理功能，从工作记忆形成、疼痛感觉到心脏起搏。 HCN 通道感知电刺激和化学刺激，并将膜兴奋性与细胞内信号通路联系起来。 HCN 通道受到电压和配体结合的双重调节，形成了蛋白质变构的绝佳研究目标。细胞内 cAMP 直接结合并打开通道。 cAMP 结合如何打开通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这个研究主题。我们通过解析 WT 和包含环核苷酸结合域 (CNBD) 的人 HCN4 C 末端片段突变形式的晶体结构，取得了重大进展。为了解决 cAMP 和整个通道之间的动态相互作用，我们建立了膜片钳荧光测定技术，可同时记录通道活动和 cAMP。我们证明 cAMP 最好与开放状态的通道结合。然后我们更进一步研究了分布式子结构域如何促进 cAMP 的全局结合。我们发现离子传导孔中的内部激活门远程控制 cAMP 结合。这一令人兴奋的发现直接涉及了如何实现配体依赖性蛋白质功能调节的基础知识。对 HCN 通道 cAMP 调节中的蛋白质变构的基本了解仍然缺乏。以下两个挑战推动我们扩大我们的研究。首先，鉴于对蛋白质分子内孤立结构域的详细了解，它们如何相互通信以及蛋白质的其余部分仍然很大程度上未知。其次，对于蛋白质变构的研究来说，整合结构和动力学的信息具有挑战性，但也至关重要。为了克服这些困难，我们建立并应用了电生理学、生物光子学、生物化学、结构和计算生物学技术。我们有三个具体目标：1）在配体-整个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 - 全通道相互作用以及重要子域（包括 S4-S5 连接子和 C 连接子）在远程影响 cAMP 结合中的作用。 2) 求解表示 cAMP 门控中过渡态的结构。我们将研究该蛋白质的未配体形式和突变形式的结构，并继续努力研究全长 HCN 结构。 3) 研究蛋白质结构和动力学之间有趣的关系。为此，我们将结合蛋白质动力学的计算和实验方法来解决分子运动，这些运动定义了 HCN 通道 cAMP 调节过程中构象变化的方向。该提案将为我们的长期研究目标奠定坚实的基础：1）以离子通道蛋白为研究平台，对蛋白质变构和蛋白质折叠有基本的了解~2）对离子治疗的见解通道相关的神经和心脏疾病。