Structure/Function characterization of μ-conotoxin KIIIA, an analgesic, nearly irreversible blocker of mammalian neuronal sodium channels

Peptide neurotoxins from one snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel nonopioid analgesics, such as subtype-selective sodium channel blockers. mu-Conotoxin KIIIA is representative of mu-conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesin activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only similar to 20% of the TTX-resistant, sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both Na(nu)1.2 and Na(nu)1.6 were strongly blocked; within experimental wash times of 40-60 min, block was reversed very little for Na(nu)1.2 and only partially for Na(nu)1.6. Other isoforms were blocked reversibly: Na(nu)1.3 (IC50 8 mu M), Na(nu)1.5 (IC50 284 mu M), and Na(nu)1.4 (IC50 80 nM). Alanine-walk and related analogs were snythesized and tested against both Na(nu)1.2 and Na(nu)1.4; replacement of Trp-8 resulted in reversible block of Na(nu)1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of Na(nu)1.4 than of Na(nu)1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of Na(nu)1.2 and that further engineering of mu-conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.