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

Peptide neurotoxins from cone 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. μ-Conotoxin KIIIA is representative of μ-conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only ∼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 _V1.2 and Na_V1.6 were strongly blocked; within experimental wash times of 40-60 min, block was reversed very little for Na_V1.2 and only partially for Na_V1.6. Other isoforms were blocked reversibly: Na_V1.3 (IC₅₀ 8 μM), Na_V1.5 (IC₅₀ 284 μM), and Na_V1.4 (IC₅₀ 80 nM). "Alanine-walk" and related analogs were synthesized and tested against both Na_V1.2 and Na_V1.4; replacement of Trp-8 resulted in reversible block of Na_V1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of Na_V1.4 than of Na_V1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of Na_V1.2 and that further engineering of μ-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.