Pore blocking mechanisms of centipede toxin SsTx-4 on the inwardly rectifying potassium channels

Dongfang Tang; Jiahui Xu; Wenhu Bao; Fanping Xu; Jieqiong Qi; Zheni Tan; Chuanli Li; Xiaofang Luo; Xia You; Mingqiang Rong; Zhonghua Liu; Cheng Tang

doi:10.1016/j.ejphar.2024.177213

Pore blocking mechanisms of centipede toxin SsTx-4 on the inwardly rectifying potassium channels

Eur J Pharmacol. 2024 Dec 18:177213. doi: 10.1016/j.ejphar.2024.177213. Online ahead of print.

Authors

Dongfang Tang¹, Jiahui Xu², Wenhu Bao³, Fanping Xu³, Jieqiong Qi³, Zheni Tan³, Chuanli Li³, Xiaofang Luo³, Xia You⁴, Mingqiang Rong⁵, Zhonghua Liu⁶, Cheng Tang⁷

Affiliations

¹ Hunan Engineering Technology Research Center for Comprehensive Development and Utilization of Biomass Resources, College of Chemistry and Bioengineering, Hunan University of Science and Engineering, Yongzhou, China; The National and Local Joint Engineering Laboratory of Animal Peptide Drug Development, College of life sciences, Hunan Normal University, Changsha, China.
² Center for Genetics and Developmental Systems Biology, Department of Obstetrics & Gynecology, Nanfang Hospital, Southern Medical University, Guangzhou, China.
³ Hunan Engineering Technology Research Center for Comprehensive Development and Utilization of Biomass Resources, College of Chemistry and Bioengineering, Hunan University of Science and Engineering, Yongzhou, China.
⁴ The National and Local Joint Engineering Laboratory of Animal Peptide Drug Development, College of life sciences, Hunan Normal University, Changsha, China.
⁵ The National and Local Joint Engineering Laboratory of Animal Peptide Drug Development, College of life sciences, Hunan Normal University, Changsha, China. Electronic address: rongmq@hunnu.edu.cn.
⁶ The National and Local Joint Engineering Laboratory of Animal Peptide Drug Development, College of life sciences, Hunan Normal University, Changsha, China. Electronic address: liuzh@hunnu.edu.cn.
⁷ The National and Local Joint Engineering Laboratory of Animal Peptide Drug Development, College of life sciences, Hunan Normal University, Changsha, China. Electronic address: chengtang@hunnu.edu.cn.

PMID: 39706465
DOI: 10.1016/j.ejphar.2024.177213

Abstract

The peptide toxin SsTx-4 derived from venom of centipede Scolopendra subspinipes mutilans was characterized as a potent antagonist of the inwardly rectifying potassium (Kir) channel subtypes Kir1.1, Kir4.1, and Kir6.2 in our previous study. Alanine-scanning mutagenesis analysis identified key molecular determinants on the SsTx-4 toxin interacting with these Kir channels, as well as those on the Kir6.2 channel interacting with the toxin. However, the key residues on Kir1.1 and Kir4.1 channels responsible for binding SsTx-4 remain unclear. Here, using a combination of site-directed mutagenesis, patch-clamp analysis, molecular docking with AlphaFold 3, and molecular dynamic simulations, we revealed that SsTx-4 acted on the Kir channels as a pore blocker, with K13 on toxin serving as the functional pore-blocking residue and other residues on it contributing to stabilize the toxin-channel complex by binding to multiple residues on the wall of the channels' outer vestibule, involving E104 on Kir1.1; D100, L115, and F133 on Kir4.1; and E108, S113, H115, and M137 on Kir6.2. Collectively, these findings advanced our understanding on the interaction between Kir channels and this prototype Kir antagonist, providing insights that could inspire the development of more potent and specific Kir subtype blockers in the future.

Keywords: Kir channels; SsTx-4; action mechanisms; pore blocker.