Structure/function characterization of micro-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 non-opioid analgesics, such as subtype-selective sodium channel blockers. Micro-conotoxin KIIIA is representative of micro-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 approximately 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(V)1.2 and Na(V)1.6 were strongly blocked; within experimental wash times of 40-60 min, block was reversed very little for Na(V)1.2 and only partially for Na(V)1.6. Other isoforms were blocked reversibly: Na(V)1.3 (IC50 8 microM), Na(V)1.5 (IC50 284 microM), and Na(V)1.4 (IC50 80 nM). "Alanine-walk" and related analogs were synthesized and tested against both Na(V)1.2 and Na(V)1.4; replacement of Trp-8 resulted in reversible block of Na(V)1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of Na(V)1.4 than of Na(V)1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of Na(V)1.2 and that further engineering of micro-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.
Zhang
Min-Min
MM
Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.
Green
Brad R
BR
Catlin
Philip
P
Fiedler
Brian
B
Azam
Layla
L
Chadwick
Ashley
A
Terlau
Heinrich
H
McArthur
Jeff R
JR
French
Robert J
RJ
Gulyas
Josef
J
Rivier
Jean E
JE
Smith
Brian J
BJ
Norton
Raymond S
RS
Olivera
Baldomero M
BM
Yoshikami
Doju
D
Bulaj
Grzegorz
G
D019943:Amino Acid Substitution; D018712:Analgesics, Non-Narcotic / Q000494:pharmacology*; D000818:Animals; D020916:Conotoxins / Q000235:genetics / Q000494:pharmacology*; D005727:Ganglia, Spinal / Q000378:metabolism / Q000473:pathology; D051379:Mice; D020125:Mutation, Missense; D009474:Neurons / Q000378:metabolism* / Q000473:pathology; D009865:Oocytes; D010146:Pain / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D010455:Peptides / Q000235:genetics / Q000494:pharmacology*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D026941:Sodium Channel Blockers / Q000494:pharmacology*; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D018712:Analgesics, Non-Narcotic; D020916:Conotoxins; D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D026941:Sodium Channel Blockers; D015222:Sodium Channels; C502265:mu-conotoxin KIIIA, Conus kinoshitai