Direct interaction of eag domains and cyclic nucleotide-binding homology domains regulate deactivation gating in hERG channels

• Published in: The Journal of general physiology 2013-10, Vol.142 (4), p.351-366
• Main Authors: Gianulis, Elena C, Liu, Qiangni, Trudeau, Matthew C
• Format: Article
• Language: English
• Subjects: Alanine; Binding Sites; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels - chemistry; Ether-A-Go-Go Potassium Channels - genetics; Ether-A-Go-Go Potassium Channels - metabolism; Fluorescence Resonance Energy Transfer; Genes; HEK293 Cells; Humans; Ion Channel Gating; Mutation; Nucleotides, Cyclic - metabolism; Potassium; Protein Structure, Tertiary; Proteins; Sequence Homology, Amino Acid
• ISSN: 0022-1295; 1540-7748
• DOI: 10.1085/jgp.201310995

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.