Identifying Novel Isoform-Specific HCN Channel Ligands using SPR

Abstract

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are expressed abundantly in the heart and central nervous system where they control cardiac and neuronal rhythmicity. Four HCN channel isoforms exist in mammals, HCN1-4. While HCN2 is the most abundantly expressed isoform in the brain, HCN4 is the predominant isoform in the heart. Identification of isoform specific regulators of HCN channels is crucial for the development of drugs that can treat cardiovascular diseases without affecting the neuronal functions and vice versa. HCN channels consist of four subunits, each containing six transmembrane segments. In addition, HCN channel subunits contain a cyclic nucleotide-binding (CNB) domain in their C-terminal region, which is linked to the pore-forming transmembrane segment by a C-linker. Upon cyclic nucleotide binding to the CNB domain, the HCN channel undergoes a conformational change that facilitates channel pore opening. The role of CNB domain in regulating channel opening via ligand binding makes it an ideal target for isoform-specific small molecule regulators. To identify isoform specific binders of HCN channel CNB domains, we conducted a surface plasmon resonance (SPR)-based screen. For the SPR screen, the C-linker/CNB domains of HCN2 and HCN4 channels were expressed in BL21 cells and purified using Ni-affinity and size-exclusion chromatography. Then the isolated C-linker/CNB domains were immobilized on the SPR senor chip, and screened for binding to the small molecules in the Spectrum Library using SPR. Using this approach, we identified nine compounds that selectively bind to the CNB domain of HCN4, but not to HCN2 channels. These results indicate that CNB domains of HCN channels harbor binding sites for HCN isoform-specific small molecule ligands.