Molecular biophysics of cAMP regulation in HCN channels

描述（由申请人提供）：HCN通道在大脑和心脏中发挥重要的生理功能，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传导途径连接起来。HCN通道受电压和配体结合的双重调控，是研究蛋白质变构的理想靶点。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难以捉摸。我们通过以下的结构，动力学和功能的研究主题接近这个研究课题。我们已经通过解决WT和突变形式的人HCN 4 C-末端片段的晶体结构取得了重大进展，所述突变形式的人HCN 4 C-末端片段包含环核苷酸结合结构域（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技术，提供通道活动和cAMP的同时记录。我们证明了cAMP优选地在开放状态下与通道结合。然后，我们进一步研究了分布式子域如何有助于cAMP的全局结合。我们发现，在离子传导孔的内部激活门远程控制cAMP结合。这一令人兴奋的发现直接触及了如何实现蛋白质功能的配体依赖性调节的基础。 对cAMP调节HCN通道的蛋白质变构的基本认识仍然缺乏。 以下两个挑战推动我们扩大研究。 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是未知的。 第二，对于蛋白质变构的研究，如何将结构和动力学的信息结合起来是一个具有挑战性的问题。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-全蛋白相互作用水平上解释变构配体调控。我们将研究其他HCN亚型中的动态cAMP -全通道相互作用以及重要亚结构域（包括S4-S5接头和C-接头）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非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的有趣关系。为此，我们将结合联合收割机的蛋白质动力学的计算和实验方法来解决的分子运动，定义的构象变化的方向在cAMP的HCN通道的调节。 这一建议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认识; 2）对离子通道蛋白的治疗有深刻的认识。 通道相关的神经和心脏疾病。

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.