The effect of the pore-blocking peptides charybdotoxin and margatoxin, both scorpion toxins, on currents through human voltage-gated hK(v)1.3 wild-type and hK(v)1.3_H399N mutant potassium channels was characterized by the whole-cell patch clamp technique. In the mutant channels, both toxins hardly blocked current through the channels, although they did prevent C-type inactivation by slowing down the current decay, during depolarization. Molecular dynamics simulations suggested that the fast current decay in the mutant channel was a consequence of amino acid reorientations behind the selectivity filter and indicated that the rigidity-flexibility in that region played a key role in its interactions with scorpion toxins. A channel with a slightly more flexible selectivity filter region exhibits distinct interactions with scorpion toxins. Our studies suggest that the toxin-channel interactions might partially restore rigidity in the selectivity filter and thereby prevent the structural rearrangements associated with C-type inactivation.