Mu-conotoxin mu-KIIIA, from Conus kinoshitai, blocks mammalian neuronal voltage-gated sodium channels (VGSCs) and is a potent analgesic following systemic administration in mice. We have determined its solution structure using NMR spectroscopy. Key residues identified previously as being important for activity against VGSCs (Lys7, Trp8, Arg10, Asp11, His12, and Arg14) all reside on an alpha-helix with the exception of Arg14. To further probe structure-activity relationships of this toxin against VGSC subtypes, we have characterized the analogue mu-KIIIA[C1A,C9A], in which the Cys residues involved in one of the three disulfides in mu-KIIIA were replaced with Ala. Its structure is quite similar to that of mu-KIIIA, indicating that the Cys1-Cys9 disulfide bond could be removed without any significant distortion of the alpha-helix bearing the key residues. Consistent with this, mu-KIIIA[C1A,C9A] retained activity against VGSCs, with its rank order of potency being essentially the same as that of mu-KIIIA, namely, Na(V)1.2 > Na(V)1.4 > Na(V)1.7 >or= Na(V)1.1 > Na(V)1.3 > Na(V)1.5. Kinetics of block were obtained for Na(V)1.2, Na(V)1.4, and Na(V)1.7, and in each case, both k(on) and k(off) values of mu-KIIIA[C1A,C9A] were larger than those of mu-KIIIA. Our results show that the key residues for VGSC binding lie mostly on an alpha-helix and that the first disulfide bond can be removed without significantly affecting the structure of this helix, although the modification accelerates the on and off rates of the peptide against all tested VGSC subtypes. These findings lay the groundwork for the design of minimized peptides and helical mimetics as novel analgesics.