title: Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating.
abstract: A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG) potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the presence of the recombinant domain is not observed with channels carrying a specific Y542C point mutation in the S4-S5 linker. On the other hand, mutations in some initial positions of the recombinant fragment also impair its ability to restore normal deactivation. Fluorescence resonance energy transfer (FRET) analysis of fluorophore-tagged proteins under total internal reflection fluorescence (TIRF) conditions revealed a substantial level of FRET between the introduced N-terminal eag fragments and the eag domain-deleted channels expressed at the membrane, but not between the recombinant eag domain and full-length channels with an intact amino terminus. The FRET signals were also minimized when the recombinant eag fragments carried single point mutations in the initial portion of their amino end, and when Y542C mutated channels were used. These data suggest that the restoration of normal deactivation gating by the N-terminal recombinant eag fragment is an intrinsic effect of this domain directed by the interaction of its N-terminal segment with the gating machinery, likely at the level of the S4-S5 linker.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oviedo, Oviedo, Spain.
authors.forename: Jorge
authors.initials: J
authors.lastname: Fernández-Trillo
authors.forename: Francisco
authors.initials: F
authors.lastname: Barros
authors.forename: Angeles
authors.initials: A
authors.lastname: Machín
authors.forename: Luis
authors.initials: L
authors.lastname: Carretero
authors.forename: Pedro
authors.initials: P
authors.lastname: Domínguez
authors.forename: Pilar
authors.initials: P
authors.lastname: de la Peña
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004594:Electrophysiology; D051638:Ether-A-Go-Go Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D017354:Point Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary
chemical_list: D051638:Ether-A-Go-Go Potassium Channels