Relating the binding of single ligands to activation in single HCN and CNG channels
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.
Dr. Lelle, Marco
Dr. Thon, Susanne
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