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

CNG 和 HCN 通道中的配体结合和激活门控：结合光学、电生理学和数学分析

• 批准号: 54760699
• 负责人: Professor Dr. Klaus Benndorf
• 金额: --
• 依托单位: Institut für Physiologie II - Herz-Kreislauf-Physiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2007
• 资助国家: 德国
• 起止时间: 2006-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/54760699?language=en
• 关键词: Ligand; binding; activation; gating; CNG

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.

环核苷酸与CNG和HCN通道的四个亚基中的每一个处的环核苷酸结合结构域的结合分别导致通道活化和调节。这种激活过程是高度合作的，即亚基彼此相互作用。最令人惊讶的是，这种协同性既有积极的一面，也有消极的一面。在这个项目中，将进一步研究这种令人惊讶的协同性的本质。与第一阶段的资助一样，实验方法结合了电生理学、光学、分子生物学和数学方法。该项目的主要目的是：（1）对于同源四聚体CNGA 2和HCN 2通道以及异源四聚体CNGA 2/CNGA 4/CNGB 1b通道，在第一个配体结合后，应确定配体与三个空亚基结合事件的顺序。该策略将基于所有三种类型的通道与四聚体多联体的成功的初步实验。这些多联体将被系统地构建和它们的结合和激活特性将被研究。(2)配体结合和激活以及解结合和失活的时间依赖性信号将通过马尔可夫模型的全局拟合策略进行分析。(3)这些研究将通过对HCN 2和异源四聚体CNGA 2/CNGA 4/CNGB 1b通道的单通道实验来补充。(4)对于HCN 2通道，将通过全局拟合策略研究协作亚基作用的性质，也用于电压依赖性门控。总之，这些结果预计将显着提高我们的四聚体CNG和HCN通道的激活过程的理解，更一般地说，多聚体变构蛋白的作用。