Gating-modifying peptide toxins preferentially bind to the voltage-sensing domain KV4.3 voltage-gated potassium channels, and the molecular determinant of this interaction remains unclear. Through unconstrained multiscale molecular dynamics simulations, we could recapitulate spontaneous binding of a gating-modifying toxin, such as SNX-482 (from tarantula Hysterocrates gigas) to the KV4.3 potassium channel at the membrane interface, overcoming the limitations of traditional docking methods. This approach revealed two likely binding poses centered on the S3-S4 linker of KV4.3, one of which included pore region residues. By replacing residues in the modeled binding poses and quantifying SNX-482 (1 μM) effects on the voltage dependence of activation of KV4.3 channels expressed in Xenopus oocytes, we determined that only the S3-S4 linker is necessary to retain SNX-482 binding. Notably, we identified M276 from the S3-S4 linker as a key stabilizing element of SNX-482/KV4.3 complex, and M276A substitution ablates toxin sensitivity. Overall, our study establishes a framework for rational drug design whose target sites lie at the membrane interface, such as gating modifiers of A-type potassium channels, relevant to neurological and cardiovascular diseases.