Biochemical characterization and nuclear magnetic resonance structure of novel alpha-conotoxins isolated from the venom of Conus consors

TWO novel alpha-conotoxins were purified and characterized from the venom o f the fish-hunting cone snail Conus consors. These peptides were identified by screening HPLC fractions of the crude venom and by binding experiments with Torpedo nicotinic acetylcholine receptor. The toxins named alpha-CnIA and alpha-CnIB exhibited sequences of 14 and 12 amino acids, respectively. The alpha-CnIA represents the main or-conotoxin contained in the venom, whe reas alpha-CnIB is present in a relatively small amount. Chemical synthesis of alpha-CnIA was carried out using the Fmoc methodology by selective disu lfide bond formation. The biological activity of the toxin was assessed in fish and mice. The alpha-CnIA inhibited the fixation of iodinated alpha-bun garotoxin to Torpedo nicotinic acetylcholine receptors with an IC50 of 0.19 mu M which can be compared to the IC50 of 0.31 mu M found for the previous ly characterized alpha-MI isolated from the piscivorous Conus magus. The sy nthetic alpha-CnIA blocked spontaneous and evoked synaptic potentials in fr og and mouse isolated neuromuscular preparations at sub-micromolar concentr ations. Solution NMR of this toxin indicated a conformational heterogeneity with the existence of different conformers in solution, at slow and interm ediate exchange rates relative to the NMR chemical shift time scale, simila r to that reported for alpha-GI and alpha-MI. NMR structures were calculate d for the major NMR signals representing more than 80% of the population at 5 degrees C.