Relating the binding of single ligands to activation in single HCN and CNG channels

Project Description

Though cyclic nucleotide-gated (CNG) and hyperpolarization activated cyclic nucleotide-modulated (HCN) ion channels belong to the same superfamily of tetrameric channels, their function is notably different: CNG channels evoke receptor potentials in vision and olfaction whereas HCN channels serve as electrical pacemaker in special neurons and cardiomyocytes. Structurally, a channel subunit contains six transmembrane helices, including a voltage-sensor domain and a pore domain as well as a cyclic nucleotide-binding domain (CNBD) that is embedded in the C-terminus. When cyclic nucleotides bind to the CNBDs, both types of channels are activated, though HCN channels require as primary activating stimulus a hyperpolarizing membrane voltage. The molecular mechanism underlying the transmission of the ligand binding to the activation gating in these channels is still poorly understood. In particular, the affinity of the four subunits for the cyclic nucleotide and the molecular interaction of the four subunits are still a mystery.

By synthesizing and using potent and efficient fluorescent cyclic nucleotides (fcGMP, fcAMP), we developed in the past decade the method of confocal patch-clamp fluorometry (cPCF) to analyze ligand binding and activation gating of HCN and CNG channels in inside-out macropatches in parallel. Our data uncovered a surprising complex interaction of the subunits for both HCN2 and CNGA2 channels, including both positive and negative cooperativity of the subunits. In this project we plan to use our fluorescent cyclic nucleotides to analyze ligand binding and activation gating at the single-molecule level. The first strategy will be to break down the cPCF to the single-molecule level, i.e. to generate patches with only one channel and to record optically ligand binding and electrically activation gating. It is expected that we can count the number of ligands bound to a channel and relate this number to the single-channel activity. These experiments will be performed in both inside-out and outside-out patches with a mechanically minimized solution volume of less than 10 attolittres containing a few ligand molecules.

The second strategy will use the TIRF-technology in supported native membranes and the number of fluorescent fcAMP or fcGMP molecules bound to single channels as function of their concentration in solution will be counted, either in isolated membrane fragments or in membranes as part of intact cells. In the latter case the effect of membrane voltage on the binding of the individual ligands will be determined. Finally, the spatial distribution of single HCN channels will be analyzed at heterologous expression in HEK293 cells and in beating mouse embryonic stem cells. The synthesis of new fluorescent ligands with elevated affinity will be a further essential part of the project. Together, our single-molecule approach is expected to provide insight into the complex and cooperative ligand binding to the four subunits in HCN and CNG channels.

Title Year Authors Journal Links
All four subunits of HCN2 channels contribute to the activation gating in an additive but intricate manner 2018 Sunkara, M.R., Schwabe, T., Ehrlich, G., Kusch, J., and Benndorf, K. JGP More
Activation gating in HCN2 channels 2018 Hummert, S., Thon, S., Eick, T., Schmauder, R., Schulz, E., and Benndorf, K. PLoS Comput Biol More
Hydrophobic alkyl chains substituted to the 8-position of cyclic nucleotides enhance activation of CNG and HCN channels by an intricate enthalpy – entropy compensation 2018 Otte, M., Schweinitz, A., Bonus, M., Enke, U., Schumann, C., Gohlke, H., and Benndorf, K. Sci Rep More
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
Deciphering the function of the CNGB1b subunit in olfactory CNG channels 2016 Nache, V., Wongsamitkul, N., Kusch, J., Zimmer, T., Schwede, F., and Benndorf, K. Sci Rep More
Family of prokaryote cyclic nucleotide-modulated ion channels 2014 Brams, M., Kusch, J., Spurny, R., Benndorf, K., and Ulens, C. Proc Natl Acad Sci USA More
Elementary functional properties of single HCN2 channels 2013 Thon, S., Schmauder, R. and Benndorf, K. Biophys J More
Hysteresis of ligand binding in CNGA2 ion channels 2013 Nache, V., Eick, T., Schulz E., Schmauder R., and Benndorf, K. Nat Commun More
Unraveling subunit cooperativity in homotetrameric HCN2 channels 2012 Benndorf, K., Thon, S., and Schulz, E. Biophys J 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
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
Low noise recording 1995 Benndorf, K. Single-Channel Reocording, 2nd edition, Springer, eds. B. Sakmann & E. Neher More