Molecular biophysics of cAMP regulation in HCN channels

HCN 通道中 cAMP 调节的分子生物物理学

• 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
• 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

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（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，从工作记忆的形成、痛觉到心律的形成。HCN通道感知电和化学刺激，并将膜兴奋性与细胞内信号通路连接起来。HCN通道受电压和配体结合的双重调控，是蛋白质变构研究的理想靶点。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是难以捉摸的。我们遵循结构、动态和功能的研究主题来探讨这个研究课题。我们通过解决WT和人类HCN4 C-末端片段的突变形式的晶体结构取得了重大进展，其中包含环核苷酸结合域（CNBD）。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，该技术可以同时记录通道活性和cAMP。我们证明了cAMP在开放状态下更容易与通道结合。然后我们进一步研究了分布子结构域如何促进cAMP的全局结合。我们发现离子传导孔内的激活门远程控制cAMP的结合。这一令人兴奋的发现直接触及了依赖配体的蛋白质功能调节是如何实现的最基本的问题。对cAMP调控HCN通道中蛋白质变构的基本理解仍然缺失。以下两个挑战推动我们扩大研究。首先，考虑到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在很大程度上仍然未知。其次，在蛋白质变构的研究中，如何将结构和动力学的信息结合起来是一项具有挑战性和关键的工作。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、结构和计算生物学等技术。我们有三个具体的目标：1)在配体-全蛋白相互作用的水平上解释变构配体调控。我们将研究其他HCN亚型中cAMP -全通道的动态相互作用，以及重要子结构域（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cAMP门控中代表过渡状态的结构。我们将继续研究该蛋白的无配体结构和突变体结构，并继续努力研究全长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来解决cAMP调节HCN通道过程中确定构象变化方向的分子运动。这一提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，利用离子通道蛋白作为研究平台；2)对离子治疗的见解