The functional surfaces of scorpion toxins representing all major pharmacological groups (anti-mammalian α,α-anti-insect,α-like,anti-mammalian β,anti-insect depressant and anti-insect-excitatory) were elucidated by mutagenesis followed by toxicity,binding,electrophysiological,and structural analyses.Despite the variations in specificity for different sodium channel,all these toxins share a common βαββ core,which was used for either swap of bioactive surfaces or restriction of activity to an individual channel subtype.Mutant double-cycle analysis of α and β-toxins against the rat brain and skeletal muscle sodium channels unraveled channel regions involved in constitution of the receptor binding sites of these toxins,and provided experimental evidence that the inter-domain spatial organization of mammalian sodium channel resembles that of voltage-gated potassium channels.Truncation of the N-and C-termini of two pharmacologically distinct β-toxins diminished their binding and typical effects,but unexpectedly exposed a novel surface of interaction with the sodium channel.The interaction of the truncated toxins with this unknown binding site could be monitored only by allosteric modulation of the binding and effects of α-toxins.The pharmacological features of this unprecedented bioactive surface provide a clue as to the evolution of such peptide toxins from smaller ancestors composed of the βαββ scaffold.