Besides classical scorpion toxin-potassium channel binding modes, novel modes remain unknown. Here, we report a novel binding mode of native toxin BmKTX towards K(v)1.3 channel. The combined experimental and computational data indicated that BmKTX-D33H analog used the classical anti-parallel beta-sheet domain as the channel-interacting interface together with the conserved channel pore-blocking Lys(26). However, the wild-type BmKTX was found to use Arg(23) rather than Lys26 as the new pore-blocking residue, and mainly adopt the turn motif between the alpha-helix and antiparallel beta-sheet domains to recognize K(v)1.3 channel. Together, these findings not only reveal that scorpion toxin-potassium channel interaction modes are more diverse than thought, but also highlight the functional role of toxin acidic residues in mediating diverse toxin-potassium channel binding modes. (C) 2014 Elsevier Inc. All rights reserved.