Mechanism of hERG inhibition by gating-modifier toxin, APETx1, deduced by functional characterization

Author:

Matsumura Kazuki,Shimomura Takushi,Kubo Yoshihiro,Oka Takayuki,Kobayashi Naohiro,Imai Shunsuke,Yanase Naomi,Akimoto Madoka,Fukuda Masahiro,Yokogawa Mariko,Ikeda Kazuyoshi,Kurita Jun-ichi,Nishimura Yoshifumi,Shimada Ichio,Osawa MasanoriORCID

Abstract

Abstract Background Human ether-à-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. APETx1 is a 42-residue peptide toxin of sea anemone Anthopleura elegantissima and inhibits hERG by stabilizing the resting state. A previous study that conducted cysteine-scanning analysis of hERG identified two residues in the S3-S4 region of the VSD that play important roles in hERG inhibition by APETx1. However, mutational analysis of APETx1 could not be conducted as only natural resources have been available until now. Therefore, it remains unclear where and how APETx1 interacts with the VSD in the resting state. Results We established a method for preparing recombinant APETx1 and determined the NMR structure of the recombinant APETx1, which is structurally equivalent to the natural product. Electrophysiological analyses using wild type and mutants of APETx1 and hERG revealed that their hydrophobic residues, F15, Y32, F33, and L34, in APETx1, and F508 and I521 in hERG, in addition to a previously reported acidic hERG residue, E518, play key roles in the inhibition of hERG by APETx1. Our hypothetical docking models of the APETx1-VSD complex satisfied the results of mutational analysis. Conclusions The present study identified the key residues of APETx1 and hERG that are involved in hERG inhibition by APETx1. These results would help advance understanding of the inhibitory mechanism of APETx1, which could provide a structural basis for designing novel ligands targeting the VSDs of KV channels.

Funder

Japan Society for the Promotion of Science KAKENHI

Vehicle Racing Commemorative Foundation

Takeda Science Foundation

Japan Agency for Medical Research and Development

Publisher

Springer Science and Business Media LLC

Subject

Cell Biology,Molecular Biology

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