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

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.