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

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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.