VanSchouwen Bryan, Akimoto Madoka, Sayadi Maryam, Fogolari Federico, Melacini Giuseppe
Departments of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario L8S 4M1, Canada.
Department of Biomedical Science and Technology, University of Udine, Piazzale Kolbe 4, 33100 Udine, Italy.
J Biol Chem. 2015 Jul 17;290(29):17642-17654. doi: 10.1074/jbc.M115.651877. Epub 2015 May 4.
The hyperpolarization-activated cyclic nucleotide-modulated (HCN) ion channels control rhythmicity in neurons and cardiomyocytes. Cyclic AMP allosterically modulates HCN through the cAMP-dependent formation of a tetrameric gating ring spanning the intracellular region (IR) of HCN, to which cAMP binds. Although the apo versus holo conformational changes of the cAMP-binding domain (CBD) have been previously mapped, only limited information is currently available on the HCN IR dynamics, which have been hypothesized to play a critical role in the cAMP-dependent gating of HCN. Here, using molecular dynamics simulations validated and complemented by experimental NMR and CD data, we comparatively analyze HCN IR dynamics in the four states of the thermodynamic cycle arising from the coupling between cAMP binding and tetramerization equilibria. This extensive set of molecular dynamics trajectories captures the active-to-inactive transition that had remained elusive for other CBDs, and it provides unprecedented insight on the role of IR dynamics in HCN autoinhibition and its release by cAMP. Specifically, the IR tetramerization domain becomes more flexible in the monomeric states, removing steric clashes that the apo-CDB structure would otherwise impose. Furthermore, the simulations reveal that the active/inactive structural transition for the apo-monomeric CBD occurs through a manifold of pathways that are more divergent than previously anticipated. Upon cAMP binding, these pathways become disallowed, pre-confining the CBD conformational ensemble to a tetramer-compatible state. This conformational confinement primes the IR for tetramerization and thus provides a model of how cAMP controls HCN channel gating.
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）通过依赖于cAMP的四聚体门控环的形成对HCN进行变构调节，该门控环跨越HCN的细胞内区域（IR），cAMP与之结合。尽管先前已绘制了cAMP结合结构域（CBD）的无配体与结合配体的构象变化，但目前关于HCN IR动力学的信息有限，据推测其在HCN的cAMP依赖性门控中起关键作用。在此，我们利用经实验核磁共振（NMR）和圆二色性（CD）数据验证和补充的分子动力学模拟，比较分析了由cAMP结合和四聚化平衡耦合产生的热力学循环四个状态下的HCN IR动力学。这一广泛的分子动力学轨迹集捕捉到了其他CBD难以捉摸的从活性状态到非活性状态的转变，并为IR动力学在HCN自抑制及其被cAMP释放中的作用提供了前所未有的见解。具体而言，IR四聚化结构域在单体状态下变得更加灵活，消除了无配体-CDB结构否则会造成的空间冲突。此外，模拟结果表明，无配体单体CBD的活性/非活性结构转变通过比先前预期更多样化的多种途径发生。cAMP结合后，这些途径被禁止，将CBD构象集合预先限制在与四聚体兼容的状态。这种构象限制使IR为四聚化做好准备，从而提供了一个cAMP如何控制HCN通道门控的模型。
