In project P2, we study the function of the C-linker and the cyclic nucleotide binding domain (CNBD) in mammalian HCN (hyperpolarization-activated and cyclic nucleotide modulated) channels. They are composed of four subunits, with the C-terminus of each subunit containing a cyclic nucleotide binding domain (CNBD) that is linked to the last transmembrane S6 helix via the C-linker (CL). Confocal patch-clamp fluorometry (cPCF), a technique which combines electrophysiological approaches and confocal fluorescence microscopy will be applied in two variations:
- to monitor activation state-dependent binding of the fluorescent cAMP derivative fcAMP and
- to analyze activation-induced conformational changes of the CL and CNBD employing the intrinsically fluorescent unnatural amino acid Anap and fluorescent dyes coupled to the channel via click chemistry.
The data will be interpreted kinetically by complex Markovian models and by MD simulations in collaboration with Holger Gohlke (University Düsseldorf). Frank Noé (Freie Universität Berlin) will provide novel strategies to fit Markovian models to the data.
Dr. Jana Kusch
Phone: 03641 - 9 397668
Prof. Dr. Klaus Benndorf
Phone: 03641 - 9 397651