Investigating Ligand Binding to HCN Channels by Surface Plasmon Resonance

Abstract

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucleotide-binding (CNB) domain in their C-terminal region linked to the pore-forming transmembrane segment with a C-linker. The C-linker couples the conformational changes caused by the direct binding of cyclic nucleotides in the CNB domain to the pore opening. Surface plasmon resonance (SPR) is a powerful biophysical tool for quantitatively investigating ligand-protein and protein-protein interactions. Here we used SPR to detect ligand binding to the isolated C-linker/CNB domain of HCN channels. The isolated C-linker/CNB domains of wild-type (WT) and L586W mutant HCN2 channels were immobilized on a NTA sensor chip and cAMP was injected over the protein coated surface. The mutant C-linker/CNB domain has been used before for ligand binding studies based on the changes in the fluorescence of the introduced tryptophan upon ligand binding. From the cyclic nucleotide concentration dependent SPR responses, we determined the binding affinity for cAMP to be 6.3 + 2.6 μM for the WT and 10.8 + 2.3μM for the mutant C-linker/CNB domains. The binding affinity determined for the mutant L586W C-linker-CNB domains determined with SPR is in agreement with the binding affinity of 13 + 2 μM determined with the fluorescence-based method. These results indicate that SPR is well suited for the detection of binding of known HCN channel ligands. Therefore, SPR can be used to identify novel HCN channel regulators for treatment of diseases associated with abnormal functions of these channels, such as epilepsy and cardiac arrhythmias.