Discovery of a heme-binding domain in a neuronal voltage-gated potassium channel

The EAG (ether-à-go-go) family of voltage-gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10–12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per–ARNT–Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.

Chlorpromazine binding to the PAS domains uncovers the effect of ligand modulation on EAG channel activity

Ether-a-go-go (EAG) potassium selective channels are major regulators of neuronal excitability and cancer progression. EAG channels contain a Per–Arnt–Sim (PAS) domain in their intracellular N-terminal region. The PAS domain is structurally similar to the PAS domains in non-ion channel proteins, where these domains frequently function as ligand-binding domains. Despite the structural similarity, it is not known whether the PAS domain can regulate EAG channel function via ligand binding. Here, using surface plasmon resonance, tryptophan fluorescence, and analysis of EAG currents recorded in Xenopus laevis oocytes, we show that a small molecule chlorpromazine (CH), widely used as an antipsychotic medication, binds to the isolated PAS domain of EAG channels and inhibits currents from these channels. Mutant EAG channels that lack the PAS domain show significantly lower inhibition by CH, suggesting that CH affects currents from EAG channels directly through the binding to the PAS domain. Our study lends support to the hypothesis that there are previously unaccounted steps in EAG channel gating that could be activated by ligand binding to the PAS domain. This has broad implications for understanding gating mechanisms of EAG and related ERG and ELK K+ channels and places the PAS domain as a new target for drug discovery in EAG and related channels. Up-regulation of EAG channel activity is linked to cancer and neurological disorders. Our study raises the possibility of repurposing the antipsychotic drug chlorpromazine for treatment of neurological disorders and cancer.