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]ο (from 2 to 98 mM) does not affect ErgTx potency, and throughsolution 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 (ΔΔG > 2 kcal/mol). We propose that the long S5-P linker of the HERG channel forms an amphipathic α-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.