Disinhibition and inhibition of HCN2 channel function by ligand binding to the cyclic nucleotide binding domain

通过配体与环核苷酸结合域结合对 HCN2 通道功能的去抑制和抑制

• 批准号: 329462230
• 负责人: Professor Dr. Holger Gohlke
• 金额: --
• 依托单位: Institut für Pharmazeutische und Medizinische Chemie
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/329462230?language=en
• 关键词: Disinhibition; inhibition; HCN2; channel; function

Hyperpolarization activated cyclic nucleotide modulated (HCN) channels are relevant disease factors, are activated by cAMP and cGMP, and consist of four subunits. Modulation of the channel function is conferred by the C-terminal region containing a cyclic nucleotide binding domain (CNBD) and a C linker (CL) region. We address the central question how changes in the conformational dynamics and energetics of the tetrameric CL-CNBD upon cyclic nucleotide (cNMP) binding relate to the ligand dependent channel gating, focusing on mammalian HCN2. We intend to answer this question at the atomistic level by molecular simulations and modelling, in close connection with experimental data from project P2, exploiting those full length structures of hHCN1 and hHCN4 have recently become available. In the first funding period, we I) characterized novel cAMP and cGMP derivatives substituted at N8 by either hydrophobic alkyl chains or similar-sized more hydrophilic heteroalkyl chains and suggested an intricate enthalpy - entropy compensation underlying the higher apparent affinity of the derivatives with the longer alkyl chains, II) tailored a series of novel fluorescent cAMP or cGMP derivatives by attaching dyes via alkyl linkers to N8, III) demonstrated that N6 modified cAMP derivatives that activate protein kinase A also act as full agonists of murine HCN2 channels, IV) developed an ensemble- and rigidity theory-based perturbation approach to analyse dynamic allostery and V) applied it to a membrane-bound transporter, receptor, and HCN2. As to the latter, predicted pathways are indicative of the influence of altered structural dynamics on allosteric signalling among four CL-CNBD subunits and, thus, provide a first atomistic interpretation of intersubunit cooperativity in HCN2 channels. In further, preliminary work, we VI) functionally and structurally characterized interactions between opposing subunits in HCN2 and VII) probed the impact of uncoupling the CL-CNBD from the transmembrane core by glycine insertions. We these results at hand, in the next funding period, we will scrutinize I) how interactions between opposing subunits impact activation-dependent CL-CNBD rotation as well as II) allosteric coupling between the “opposing subunit region” and the cNMP binding site, III) investigate signal transmission between the CL-CNBD and the transmembrane core in variants with a defined number of uncoupled and/or functional CL-CNBDs, IV) probe the role of helices D and E on cAMP affinity and allosteric signal transmission, V) develop selective HCN agonists and identify inhibitors that stabilize the inactive state of the CL-CNBD, and, if time permits, VI) generate a structural model of HCN2 with dilated channel structure. These studies will significantly advance our understanding of how structural dynamics at the atomistic level and disinhibition and inhibition of HCN2 channel function are coupled by ligand binding to the CL-CNBD.

超极化激活的环核苷酸调节（HCN）通道是相关的疾病因素，由cAMP和cGMP激活，并由四个亚基组成。通道功能的调节由含有环核苷酸结合结构域（CNBD）和C接头（CL）区的C末端区赋予。我们解决的中心问题是如何在构象动力学和能量的四聚体的CL-CNBD环核苷酸（cNMP）结合后的变化涉及到配体依赖性通道门控，专注于哺乳动物HCN 2。我们打算通过分子模拟和建模在原子水平上回答这个问题，与项目P2的实验数据密切相关，利用最近可用的hHCN 1和hHCN 4的全长结构。在第一个资助期内，我们I）表征了在N8处被疏水烷基链或类似大小的更亲水的杂烷基链取代的新型cAMP和cGMP衍生物，并提出了复杂的焓-熵补偿，这是衍生物与较长烷基链的更高表观亲和力的基础，II）通过经由烷基接头将染料连接到N8来定制一系列新型荧光cAMP或cGMP衍生物，III）证明了活化蛋白激酶A的N6修饰的cAMP衍生物也作为鼠HCN 2通道的完全激动剂，IV）开发了基于系综和刚性理论的扰动方法来分析动态变构，V）将其应用于膜结合转运蛋白、受体和HCN 2。至于后者，预测的途径是指示的影响改变结构动力学的变构信号之间的四个CL-CNBD亚基，因此，提供了第一个原子的解释亚基间的协同HCN 2通道。在进一步的初步工作中，我们VI）在功能上和结构上表征了HCN 2中相对亚基之间的相互作用，VII）探索了通过甘氨酸插入使CL-CNBD与跨膜核心解偶联的影响。我们手头有这些结果，在下一个资助期，我们将仔细研究I）相对亚基之间的相互作用如何影响活化依赖性CL-CNBD旋转以及II）“相对亚基区域”和cNMP结合位点之间的变构偶联，III）研究具有限定数量的未偶联和/或功能性CL-CNBD的变体中CL-CNBD和跨膜核心之间的信号传递，IV）探测螺旋D和E对cAMP亲和力和变构信号传递的作用，V）开发选择性HCN激动剂并鉴定稳定化CL-CNBD的非活性状态的抑制剂，以及，如果时间允许，VI）产生具有扩张通道结构的HCN 2的结构模型。这些研究将显着推进我们的理解如何在原子水平上的结构动力学和去抑制和抑制HCN 2通道功能耦合配体结合的CL-CNBD。