Chlorpromazine inhibits EAG1 channels by altering the interdomain coupling.

EAG1 depolarization-activated potassium selective channels are important targets for treatment of cancer and neurological disorders. EAG1 channels are formed by a tetrameric subunit assembly with each subunit containing an N-terminal Per-Arnt-Sim (PAS) domain and C-terminal cyclic nucleotide-binding homology (CNBH) domain. The PAS and CNBH domains from adjacent subunits interact and form an intracellular tetrameric ring that regulates the EAG1 channel gating, including the movement of the voltage sensor domain (VSD) from closed to open states. Small-molecule ligands can inhibit EAG1 channels by binding to their PAS domains. However, the allosteric pathways of this inhibition are not known. Here, we combined molecular dynamics (MD) simulations, network analysis, surface plasmon resonance (SPR), and mutagenesis-coupled electrophysiology to investigate the allosteric pathways of EAG1 channel regulation by a small-molecule ligand. MD simulations and network analysis revealed that chlorpromazine, a PAS domain small-molecule binder, alters interactions between the PAS and CNBH domains. Consistent with computational predictions, chlorpromazine affected interactions between the isolated PAS and CNBH domains probed with SPR. Our network analysis also indicated that binding chlorpromazine to the PAS domain alters coupling between all major regulatory domains of EAG1, including the intracellular PAS/CNBH domain ring, VSD and pore of the channel. Interestingly, chlorpromazine binding to the PAS domain did not substantially alter Cole-Moore shift characteristic of EAG1 channels, suggesting that chlorpromazine has little effect on the VSD movement from the deep closed to opened states. This further suggests that, if chlorpromazine binding alters coupling between the PAS/CNBH domain ring, VSD, and pore, as predicted by the network analysis, these alterations occur at potential more depolarized than the ones eliciting the Cole-Moore shift. Taken together, our study provides an insight into the allosteric pathways of EAG1 channel regulation by small-molecule PAS domain binders.