VanSchouwen Bryan, Melacini Giuseppe
Department of Chemistry and Chemical Biology, McMaster University , 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.
Department of Biochemistry and Biomedical Sciences, McMaster University , Hamilton, Ontario L8S 4M1, Canada.
J Phys Chem B. 2016 Oct 27;120(42):10936-10950. doi: 10.1021/acs.jpcb.6b07735. Epub 2016 Oct 18.
The hyperpolarization-activated cyclic-nucleotide-modulated (HCN) ion channels control rhythmicity in neurons and cardiomyocytes. Cyclic AMP (cAMP) modulates HCN activity through the cAMP-dependent formation of a tetrameric gating ring spanning the intracellular region (IR) of HCN. In the absence of cAMP, the IR cAMP-binding domain (CBD) mainly samples its inactive conformation, resulting in steric clashes that destabilize the IR tetramer. Although these clashes with the inactive CBD are released through tetramer dissociation into monomers, functional mutagenesis suggests that the apo IR is not fully monomeric. To investigate the inhibitory nonmonomeric IR species, we performed molecular dynamics simulations starting from "hybrid" structures that are tetrameric but contain inactive apo-state CBD conformations. The ensemble of simulated trajectories reveals that full dissociation of the tetramer into monomers is not necessary to release the steric hindrance with the inactive CBD. Specifically, we found that partial dissociation of the tetramer into dimers is sufficient to accommodate four inactive CBDs, while reduction of the quaternary symmetry of the nondissociated tetramer from 4- to 2-fold permits accommodation of two inactive CBDs. Our findings not only rationalize available electrophysiological, fluorometry, and sedimentation equilibrium data, but also provide unprecedented structural insight into previously elusive nonmonomeric autoinhibitory HCN species.
超极化激活的环核苷酸调制（HCN）离子通道控制神经元和心肌细胞的节律性。环磷酸腺苷（cAMP）通过cAMP依赖性形成跨越HCN细胞内区域（IR）的四聚体门控环来调节HCN活性。在没有cAMP的情况下，IR cAMP结合结构域（CBD）主要处于其非活性构象，导致空间冲突，使IR四聚体不稳定。尽管与非活性CBD的这些冲突通过四聚体解离成单体而得以释放，但功能诱变表明无配体的IR并非完全单体状态。为了研究抑制性非单体IR物种，我们从“混合”结构开始进行分子动力学模拟，这些结构是四聚体，但包含非活性无配体状态的CBD构象。模拟轨迹的集合表明，四聚体完全解离成单体对于释放与非活性CBD的空间位阻并非必要。具体而言，我们发现四聚体部分解离成二聚体足以容纳四个非活性CBD，而非解离的四聚体四级对称性从4倍降至2倍则允许容纳两个非活性CBD。我们的研究结果不仅使现有的电生理学、荧光测定和沉降平衡数据合理化，还为以前难以捉摸的非单体自抑制HCN物种提供了前所未有的结构见解。
