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

超极化激活的环核苷酸调节（HCN）通道是相关的疾病因子，由cAMP和cGMP激活，由四个亚基组成。通道功能的调节是由含有环核苷酸结合域（CNBD）和C连接子（CL）区域的C端区域赋予的。我们解决了四聚体CL-CNBD在环核苷酸（cNMP）结合时的构象动力学和能量学变化如何与配体依赖性通道门控相关的核心问题，重点关注哺乳动物HCN2。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，并与P2项目的实验数据密切相关，利用最近可用的hHCN1和hHCN4的全长结构。在第一个资助期，我们I)表征了新的cAMP和cGMP衍生物在N8上被疏水烷基链或类似大小的更亲水的杂烷基链取代，并提出了复杂的焓熵补偿，表明衍生物与较长的烷基链具有更高的表观亲和力。II)通过烷基连接剂将染料附着在N8上，从而设计了一系列新的荧光cAMP或cGMP衍生物。III)证明了激活蛋白激酶A的N6修饰的cAMP衍生物也可以作为小鼠HCN2通道的完全激动剂，IV)开发了基于集合和刚性理论的微扰方法来分析动态变构，V)将其应用于膜结合转运体，受体和HCN2。对于后者，预测的通路表明了改变的结构动力学对四个CL-CNBD亚基之间变构信号传导的影响，因此，提供了HCN2通道中亚基间协同性的第一个原子解释。在进一步的初步工作中，我们（VI）在功能和结构上表征了HCN2中对立亚基之间的相互作用，（VII）探索了甘氨酸插入从跨膜核心解耦CL-CNBD的影响。根据这些结果，在下一个资助期，我们将仔细研究I)对立亚基之间的相互作用如何影响激活依赖性CL-CNBD旋转，以及II)“对立亚基区域”与cNMP结合位点之间的变弹性耦合，III)在具有一定数量的未偶联和/或功能性CL-CNBD的变体中，研究CL-CNBD与跨膜核心之间的信号传递。IV)探索螺旋D和E在cAMP亲和力和变构信号传递中的作用，V)开发选择性HCN激动剂并鉴定稳定CL-CNBD失活状态的抑制剂，如果时间允许，VI)生成具有扩张通道结构的HCN2结构模型。这些研究将极大地促进我们对原子水平结构动力学和HCN2通道功能的解除抑制和抑制是如何通过配体结合CL-CNBD耦合的理解。

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.