The hERG N-Terminal eag Domain Directly Interacts with the C-Terminal Cyclic Nucleotide-Binding Homology Domain

Abstract

Human ether-a-go-go-related gene (hERG) voltage-gated potassium channels exhibit unusually slow deactivation kinetics that are regulated by the N-terminal eag domain. The mechanism by which the eag domain regulates deactivation remains unclear. Recently we showed that the cyclic nucleotide-binding homology domain (CNBHD) in the C-terminal region is required for eag domain regulation of gating, and the two domains directly interact in a biochemical pull-down interaction assay. Here we sought to investigate the CNBHD as a putative binding site for the eag domain by using a combination of electrophysiology and Forster Resonance Energy Transfer (FRET) in HEK293 cells. Whole-cell patch clamp analysis revealed that channels lacking either the eag domain (Δeag), the CNBHD (ΔCNBHD), or both domains (Δeag ΔCNBHD) exhibited similarly fast deactivation kinetics. Co-expression of an eag domain gene fragment (N-eag) slowed deactivation in Δeag channels, but not in channels lacking a CNBHD (ΔCNBHD or Δeag ΔCNBHD). FRET analysis revealed that N-eag-CFP was in close proximity to Δeag channels tagged with a Citrine (Δeag-Citrine), but not with Δeag ΔCNBHD-Citrine channels, suggesting the CNBHD is required for the eag domain to bind to the channel. We found that co-expression of Δeag-CFP channels with ΔCNBHD-Citrine channels restored WT-like channel gating, and revealed a positive FRET signal, suggesting an intersubunit interaction between the eag domain and CNBHD. To test for a direct interaction between the eag domain and CNBHD, we utilized the FRET Two-Hybrid Peptide Hybridization Assay in which we co-expressed fluorophore-tagged eag domains and CNBHDs and measured FRET. We observed a positive and significant FRET signal between isolated eag domains and CNBHDs, suggesting a direct and specific interaction between the two domains. Together these findings suggest that eag domain directly binds the CNBHD in order to regulate channel gating.