Combinatorial Residue Recognition in SsTx-4 Inhibition of the Kir4.1/5.1 Channel

The Kir4.1/5.1 channel is predominantly expressed in renal tubular epithelial cells and glial cells of the nervous system, where it plays essential physiological roles. In our previous work, the centipede venom peptide SsTx-4 was shown to strongly inhibit the homomeric Kir4.1 channel; however, its activity toward the heteromeric Kir4.1/5.1 channel remained unclear. Here, we show that SsTx-4 inhibits the Kir4.1/5.1 channel with a potency comparable to that of Kir4.1. Electrophysiological recordings combined with site-directed mutagenesis, structural modeling, and molecular dynamics simulations demonstrate that SsTx-4 blocks the Kir4.1/5.1 channel via a conserved pore-occlusion mechanism, while utilizing a distinct, subtype-dependent combination of peptide residues for channel binding. Structural analyses indicate that K13 inserts into the pore and interacts with the selectivity filter, whereas K10 forms a hydrogen bond with Y135 of the Kir4.1 subunit and F44 forms a hydrophobic contact with Y136 of the Kir5.1 subunit. Additional residues, including K4, R12, F14, Y16, K40, and K45, further stabilize the toxin-channel complex through hydrogen bonds and hydrophobic interactions. Notably, the basic residues K4, K40, and K45 may cooperatively contribute to binding to the Kir4.1/5.1 channel. Together, these findings support a combinatorial residue recognition mechanism underlying SsTx-4 inhibition of Kir channels and provides mechanistic insights into understanding peptide-Kir channel interactions.