Perturbation of HCN1 response to small molecule modulation

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are proteins with essential roles in pacemaking activity, neural signaling, and the development of neuropathic pain. Several allosteric ligands have been shown to inhibit HCN1; however, the structural basis of their action has yet to be defined. We uncovered putative binding sites for the intravenous general anesthetic propofol (2,6-di-isopropylphenol), a known HCN1 small molecule allosteric modulator, by cryoEM. Location of the sites correlate well with computational models. Moreover, these sites align with our bioinformatically derived allosteric network for channel activation, suggesting a mode of inhibition. Perturbation of one of the structurally identified binding sites resulted in differential inhibitor response compared to wildtype HCN1, as measured by Xenopus laevis oocyte two electrode voltage clamp. Future studies will further characterize these inhibitor binding sites and mechanistically investigate the source of isoform specificity.