title: Mapping the binding site of a human ether-a-go-go-related gene-specific peptide toxin (ErgTx) to the channel's outer vestibule.
abstract: The goals of this study are to investigate the mechanism and site of action whereby a human ether-a-go-go-related gene (HERG)-specific scorpion peptide toxin, ErgTx, suppresses HERG current. We apply cysteine-scanning mutagenesis to the S5-P and P-S6 linkers of HERG and examine the resulting changes in ErgTx potency. Data are compared with the characteristics of charybdotoxin (ChTx, or its analogs) binding to the Shaker channel. ErgTx binds to the outer vestibule of HERG but may not physically occlude the pore. In contrast to ChTx. Shaker interaction, elevating [K](o) (from 2 to 98 mm) does not affect ErgTx potency, and through-solution electrostatic forces only play a minor role in influencing ErgTx.HERG interaction. Cysteine mutations of three positions in S5-P linker (Trp-585, Gly-590, and Ile-593) and 1 position in P-S6 linker (Pro-632) induce profound changes in ErgTx binding (DeltaDeltaG > 2 kcal/mol). We propose that the long S5-P linker of the HERG channel forms an amphipathic alpha-helix that, together with the P-S6 linker, forms a hydrophobic ErgTx binding site. This study paves the way for future mutant cycle analysis of interacting residues in the ErgTx.HERG complex, which, in conjunction with NMR determination of the ErgTx solution structure, will yield information about the topology of HERG's outer vestibule.
authors.affiliation: Department of Physiology, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
authors.forename: Liliana
authors.initials: L
authors.lastname: Pardo-Lopez
authors.forename: Mei
authors.initials: M
authors.lastname: Zhang
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.forename: Min
authors.initials: M
authors.lastname: Jiang
authors.forename: Lourival D
authors.initials: LD
authors.lastname: Possani
authors.forename: Gea-Ny
authors.initials: GN
authors.lastname: Tseng
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000737:chemistry* / Q000378:metabolism; D027682:Cation Transport Proteins; D018999:Charybdotoxin / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D000072237:ERG1 Potassium Channel; D004594:Electrophysiology; D051638:Ether-A-Go-Go Potassium Channels; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D011188:Potassium; D015221:Potassium Channels / Q000737:chemistry* / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated; D011485:Protein Binding; D012604:Scorpion Venoms / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051637:Shaker Superfamily of Potassium Channels; D019789:Tetraethylammonium / Q000494:pharmacology; D013997:Time Factors; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG
chemical_list: D002121:Calcium Channel Blockers; D027682:Cation Transport Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C072312:ERG protein, human; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; C092509:KCNH6 protein, human; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D012604:Scorpion Venoms; D051637:Shaker Superfamily of Potassium Channels; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; C118951:ergotoxin, Centruroides noxius; D018999:Charybdotoxin; D019789:Tetraethylammonium; D003545:Cysteine; D011188:Potassium