Elucidating assembly, ligand binding and gating of heterotetrameric CNG channels by FRET

Project Description
Principal Investigator(s)

Cyclic nucleotide-gated (CNG) channels play an important role in the visual and olfactory system. They constitute the final step of a signal transduction cascade by which light or scent are transduced into a change of the membrane potential. Native CNG channels are heterotetramers, consisting of up to three different subunits. Each subunit has six membrane-spanning segments. The binding of a cyclic nucleotide (cAMP or cGMP) to the cyclic nucleotide binding domains (CNBDs) located intracellularly at the C-terminus promotes an allosteric conformational change that is transmitted through the C-linker to the channel pore leading to channel opening.

Although all subunits contain an intracellular located cyclic nucleotide-binding domain, only the CNGA1, CNGA2 and CNGA3 are capable of forming functional homotetrameric channels when expressed in heterologous expression systems. CNGA4 and CNGB subunits only modulate channel function and control plasma membrane targeting. Direct evidence for their contribution to channel activation by ligand binding was presented up to now only for the olfactory CNGA4 subunit.

This project aims at elucidating the role of the CNGB1a and CNGB3 subunits of our visual system. Specifically, we want to find out whether these subunits bind cGMP at physiological concentrations and if so, in which sequence binding to the respective subunits occurs.

The following questions will be addressed: In which sequence do the ligands bind to the subunits? Is the opening of the channel pore the result of a concerted conformational change of all four subunits? What conformational changes are induced by ligand binding? To address these questions we will use among other methods confocal patch-clamp fluorometry (cPCF) and Förster Resonance Energy Transfer (FRET) measurements.

A second focus of the project will be on the mechanisms by which the cell ensures that channels are expressed in a fixed stoichiometry. These mechanisms are responsible for the successful channel trafficking to the plasma membrane whose malfunction was shown to be the cause of many channelopathies. By using fluorescence lifetime based FRET measurements we will investigate in which compartment the channels assemble, which subunits associate first, and how channel transport is controlled.

Title Year Authors Journal Links
Quantifying the cooperative subunit action in a multimeric membrane receptor 2016 Wongsamitkul, N., Nache V., Eick, T., Hummert, S., Schulz, E., Schmauder, R., Schirmeyer, J., Zimmer, T., and Benndorf, K. Sci. Rep. More
Hysteresis of ligand binding in CNGA2 ion channels 2013 Nache, V., Eick, T., Schulz E., Schmauder R., and Benndorf, K. Nat Commun More
Differential regulation by cyclic nucleotides of the CNGA4 and CNGB1b subunits in olfactory cyclic nucleotide-gated channels 2012 Nache, V., Zimmer, T., Wongsamitkul, N., Schmauder, R., Kusch, J., Reinhardt, L., Bonigk, W., Seifert, R., Biskup, C., Schwede, F. and Benndorf, K. Sci Signal More
How subunits cooperate in cAMP-induced activation of homotetrameric HCN2 channels 2011 Kusch, J., Thon, S., Schulz, E., Biskup, C., Nache, V., Zimmer, T., Seifert, R., Schwede, F., and Benndorf, K. Nat Chem Biol More
Interdependence of receptor activation and ligand binding in HCN2 pacemaker channels 2010 Kusch, J., Biskup, C., Thon, S., Schulz, E., Nache, V., Zimmer, T., Schwede, F. and Benndorf, K. Neuron More
Relating ligand binding to activation gating in CNGA2 channels 2007 Biskup, C., Kusch, J., Schulz, E., Nache, V., Schwede, F., Lehmann, F., Hagen, V. and Benndorf, K. Nature More
Multi-dimensional fluorescence lifetime and FRET measurements 2007 Biskup, C., Zimmer, T., Kelbauskas, L., Hoffmann, B., Klöcker, N., Becker, W., Bergmann, A., and Benndorf, K. Microsc Res Tech More
Gating of cyclic nucleotide-gated (CNGA1) channels by cGMP jumps and depolarizing voltage steps 2006 Nache, V., Kusch, J., Hagen, V., and Benndorf, K. Biophys J More
Activation of olfactory-type cyclic nucleotide-gated channels is highly cooperative 2005 Nache, V., Schulz, E., Zimmer, T., Kusch, J., Biskup, C., Koopmann, R., Hagen, V. and Benndorf, K. J Physiol More
Interaction of PSD-95 with potassium channels visualized by fluorescence lifetime-based resonance energy transfer imaging 2004 Biskup, C., Kelbauskas, L., Zimmer, T., Benndorf, K., Bergmann, A., Becker, W., Ruppersberg, J.P., Stockklausner, and C., Klöcker, N. J Biomed Opt More
FRET between cardiac Na+ channel subunits measured with a confocal microscope and a streak camera 2004 Biskup, C., Zimmer, T., and Benndorf, K. Nat Biotech More