Examining Drug Binding in HCN Channels

Abstract

Neuronal HCN channels are open when neurons are not firing action-potentials (sub-threshold potentials), and reduce the input resistance of the cell membrane making it less responsive to incoming inputs. These combined properties make them excellent targets for fine-tuning of intrinsic neuronal excitability. HCN channels are also expressed in cardiac conduction tissue where they establish the pace of the heartbeat, and enable sensitivity to autonomic simulation. Moreover, since HCN channels are not found in vascular tissue, targeted inhibition of HCN channels has strong therapeutic potential as bradycardic agents, anti-convulsants, and analgesics without adverse effects on pulmonary and vascular smooth muscle tone. While several molecules that target HCN channels have been identified, their low affinity and lack of isoform specificity prevents wide-spread use of these current HCN inhibitors. To advance the development of isoform specific inhibitors, we are using computational and electrophysiological methods to characterize the binding sites of known inhibitors and identify key interactions. Using computational docking approaches, we have examined the binding of 13 known inhibitors to the atomic structure of human HCN1 (PDB: 5U6O), and a homology model of the open state derived from the closely related eukaryotic CNG channel (PDB: 5H3O). Our results indicate that “bradine” inhibitors bind in the pore cavity with no preferred orientation, providing possible insight into their low affinity. Other results indicate that clonidine and alinidine are likely pore blockers as well, but with greater conformational restraint. Detailed characterization of the mode of binding and residues lining the binding pocket may enable the development of isoform specific inhibitors that can be used as chemical probes or therapeutic agents.