Scrutinizing the function of the cyclic-nucleotide binding domain in the activation of HCN channels

在这个项目中，我们研究了环核苷酸(CN)调节的离子通道的配体依赖的门控，从而聚焦于哺乳动物的超极化激活的环核苷酸调节的通道。HCN通道是四聚体，其中CN诱导的门控是由四个细胞内C末端结合结构域(CNBD)介导的，通过C连接物(CLS)连接到最后一个跨膜螺旋(S6)，从而也形成了四聚体结构。结合电生理学和荧光显微镜，用电生理学、诱变和膜片钳荧光法研究了细胞内通道部分的构象变化。除了研究CL和CNBD构象变化引起的环境敏感染料的离子电流和荧光变化的动力学外，还将监测荧光标记的cAMP衍生物的结合情况。这些数据将通过与H.Gohlke(P7)合作的MD模拟和扩展我们的马尔科夫建模策略，从而与F.Noé(P8)合作，对数据进行运动学解释。

In this project we study the ligand-dependent gating of cyclic nucleotide (CN)-modulated ion channels, thereby focusing on mammalian HCN (hyperpolarization-activated cyclic-nucleotide modulated) channels. HCN channels are tetramers, in which CN-induced gating is mediated by four intracellular C-terminal binding domains (CNBDs), linked to the last transmembrane helices (S6) via C-linkers (CLs), thereby also forming a tetrameric structure. Conformational changes in this intracellular channel portion are studied by electrophysiology, mutagenesis and patch-clamp fluorometry, combining electrophysiology and fluorescence microscopy. Besides studying the kinetics of ion currents and fluorescence changes of environment-sensitive dyes, caused by conformational changes of the CL and CNBD, the binding of fluorescently tagged cAMP derivatives will be monitored. The data will be interpreted kinetically by MD simulations in collaboration with H. Gohlke (P7) and by extending our Markovian modeling strategies, thereby collaborating with F. Noé (P8).