Ligand binding and activation gating in CNG and HCN channels: Combined optical, electrophysiological and mathematical analyses

环核苷酸与CNG和HCN通道四个亚基的环核苷酸结合域的结合分别导致通道的激活和调制。这种激活过程是高度合作的，即亚基之间相互作用。最令人惊讶的是，这种合作既有积极的一面，也有消极的一面。在这个项目中，我们将进一步研究这种令人惊讶的合作关系的本质。与第一期资助一样，实验方法结合了电生理学、光学、分子生物学和数学方法。该项目的主要目标是：(1)对于同质四聚体CNGA2和HCN2通道以及异四聚体CNGA2/CNGA4/CNGB1b通道，应在第一个配体结合后确定与三个空亚基的配体结合事件的顺序。该战略将基于对所有三种类型的通道进行的成功的四聚体级联器的初步实验。这些级联体将被系统地构建，并将研究它们的结合和活化性质。(2)采用马尔可夫模型的全局拟合策略，分析了配体结合和激活以及解离和失活的时间相关信号。(3)对HCN_2通道和CNGA_2/CNGA_4/CNGB1b异四聚体通道的单通道实验将补充这些研究。(4)对于HCN_2通道，协作子单元作用的性质将通过全局适配策略来研究，对于电压依赖的门控也是如此。总之，这些结果有望显著提高我们对四聚体CNG和HCN通道激活过程的理解，以及更广泛地理解多聚体变构蛋白的作用。

The binding of cyclic nucleotides to the cyclic nucleotide binding domain at each of the four subunits of CNG and HCN channels leads to channel activation and modulation, respectively. This activation process is highly cooperative, i.e. the subunits interact with each other. Most surprisingly, this cooperativity is both positive and negative. In this project the nature of this surprising cooperativity will be further investigated. As in the first term of funding, the experimental approach combines electrophysiological, optical, molecular biological, and mathematical methods. The main aims of the project are: (1) For homotetrameric CNGA2 and HCN2 channels as well as for heterotetrameric CNGA2/CNGA4/CNGB1b channels the sequence of ligand binding events to the three empty subunits shall be determined after the first ligand has been bound. The strategy will be based on successful preliminary experiments for all three types of channels with tetrameric concatamers. These concatamers will be systematically constructed and their binding and activation properties will be studied. (2) The time-dependent signals of ligand binding and activation as well as unbinding and deactivation will be analyzed by means of global fit strategies with Markovian models. (3) These investigations will be complemented by single-channel experiments on both HCN2 and heterotetrameric CNGA2/CNGA4/CNGB1b channels. (4) For HCN2 channels the nature of the cooperative subunit action will be studied by the global fit strategy also for voltage-dependent gating. Together, the results are expected to significantly improve our understanding for the activation process of tetrameric CNG and HCN channels and, more generally, for the action of multimeric allosteric proteins.