title: Direct interaction of eag domains and cyclic nucleotide-binding homology domains regulate deactivation gating in hERG channels.
abstract: Human ether-á-go-go (eag)-related gene (hERG) potassium channels play a critical role in cardiac repolarization and are characterized by unusually slow closing (deactivation) kinetics. The N-terminal "eag" domain and a C-terminal C-linker/cyclic nucleotide-binding homology domain (CNBHD) are required for regulation of slow deactivation. The region between the S4 and S5 transmembrane domains (S4-S5 linker) is also implicated in this process, but the mechanism for regulation of slow deactivation is unclear. Here, using an eag domain-deleted channel (hERG Δeag) fused to Citrine fluorescent protein, we found that most channels bearing individual alanine mutations in the S4-S5 linker were directly regulated by recombinant eag domains fused to a cyan fluorescent protein (N-eag-CFP) and had robust Förster resonance energy transfer (FRET). Additionally, a channel bearing a group of eight alanine residues in the S4-S5 linker was not measurably regulated by N-eag-CFP domains, but robust FRET was measured. These findings demonstrate that the eag domain associated with all of the S4-S5 linker mutant channels. In contrast, channels that also lacked the CNBHD (hERG Δeag ΔCNBHD-Citrine) were not measurably regulated by N-eag-CFP nor was FRET detected, suggesting that the C-linker/CNBHD was required for eag domains to directly associate with the channel. In a FRET hybridization assay, N-eag-CFP had robust FRET with a C-linker/CNBHD-Citrine, suggesting a direct and specific interaction between the eag domain and the C-linker/CNBHD. Lastly, coexpression of a hERG subunit lacking the CNBHD and the distal C-terminal region (hERG ΔpCT-Citrine) with hERG Δeag-CFP subunits had FRET and partial restoration of slow deactivation. Collectively, these findings reveal that the C-linker/CNBHD, but not the S4-S5 linker, was necessary for the eag domain to associate with the channel, that the eag domain and the C-linker/CNBHD were sufficient for a direct interaction, and that an intersubunit interaction between the eag domain and the C-linker/CNBHD regulated slow deactivation in hERG channels at the plasma membrane.
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201.
authors.forename: Elena C
authors.initials: EC
authors.lastname: Gianulis
authors.forename: Qiangni
authors.initials: Q
authors.lastname: Liu
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Trudeau
mesh_terms: D001665:Binding Sites; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D009154:Mutation; D009712:Nucleotides, Cyclic / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D009712:Nucleotides, Cyclic
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