Kazuki Matsumura1, Takushi Shimomura2, Yoshihiro Kubo2, Takayuki Oka3, Naohiro Kobayashi4,5, Shunsuke Imai6, Naomi Yanase1, Madoka Akimoto1, Masahiro Fukuda1, Mariko Yokogawa1, Kazuyoshi Ikeda1, Jun-Ichi Kurita7, Yoshifumi Nishimura7, Ichio Shimada6, Masanori Osawa8. 1. Graduate School of Pharmaceutical Sciences, Keio University, Shibakoen, Minato-ku, Tokyo, 105-8512, Japan. 2. Division of Biophysics and Neurobiology, Department of Molecular and Cellular Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki-shi, Aichi, 444-8585, Japan. 3. Nanion Technologies Japan K.K., Tokyo Laboratory, Wakamatsu-cho, Shinjuku-ku, Tokyo, 162-0056, Japan. 4. Institute for Protein Research, Osaka University, Yamadaoka, Suita-shi, Osaka, 565-0871, Japan. 5. NMR Science and Development Division, RSC, RIKEN, Suehiro-cho, Tsurumi-ku, Yokohama-shi, Kanagawa, 230-0045, Japan. 6. Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. 7. Graduate School of Medical Life Science, Yokohama City University, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan. 8. Graduate School of Pharmaceutical Sciences, Keio University, Shibakoen, Minato-ku, Tokyo, 105-8512, Japan. osawa-ms@pha.keio.ac.jp.
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.
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.
Authors: Siyi He; Mohamed Taha Moutaoufik; Saadul Islam; Amit Persad; Adam Wu; Khaled A Aly; Humphrey Fonge; Mohan Babu; Francisco S Cayabyab Journal: Biochim Biophys Acta Rev Cancer Date: 2020-03-02 Impact factor: 10.680
Authors: G C P van Zundert; J P G L M Rodrigues; M Trellet; C Schmitz; P L Kastritis; E Karaca; A S J Melquiond; M van Dijk; S J de Vries; A M J J Bonvin Journal: J Mol Biol Date: 2015-09-26 Impact factor: 5.469