Characterization and Nuclear Magnetic Resonance Structure of Novel alpha-Conotoxins
Isolated from the Venom of Conus consors.
Favreau, P., Krimm, I., Le Gall, F., Bobenrieth,
M.J., Lamthanh, H., Bouet, F., Servent, D., Molgo, J., Menez, A., Letourneux,
Y. and Lancelin, J-M. Biochemistry 38: 6317-6326 (1999).
Two novel alpha-conotoxins, alpha-CnIA
(major) and alpha-CnIB (minor), of 14 and 12 amino acids, respectively,
purified by HPLC from the venom of the fish-hunting cone snail Conus
alpha-CnIA and alpha-CnIB are very similar
in structure to alpha-conotoxin-MI (from C. magus). alpha-CnIA has a Tyr
(Y) in position 11, instead of the Asn (N) in alpha-MI. alpha-CnIA and
CnIB possess the three/five loop structure of conotoxins GI, GIA, GII,
MI, SI, SIA, and SII (alpha3/5 subclass).
These peptides were characterized by binding
experiments with Torpedo nicotinic acetylcholine receptor and by
NMR. Solution NMR indicated the existence of different conformers
in solution, similar to that reported for alpha-GI and alpha-MI. Chemical
synthesis of alpha-CnIA was achieved by Fmoc methodology and by selective
disulfide bond formation.
The biological activity of the toxin
was assessed in fish and mice. Alpha-CnIA inhibited the fixation of iodinated
alpha-bungarotoxin to Torpedo nicotinic acetylcholine receptors with an
IC50 of 0.19 uM (compared to 0.31 uM for alpha-MI from Conus magus).
Synthetic alpha-CnIA blocked spontaneous and evoked synaptic potentials
in frog and mouse isolated neuromuscular preparations at sub-micromolar
concentrations. The effects of alpha-CnIA were reversible. On an equimolar
basis alpha-CnIA was 6-8 times more potent than alpha-GI in blocking postsynaptic
nAChRs at the frog neuromuscular junction. Results obtained with a mouse
phrenic nerve hemidiaphragm preparation provide the first recordings showing
the blockade of MEPPs by an alpha-conotoxin (previous studies with mammalian
preparations have mostly relied on twitch tension recordings).
The difference in affinity of alpha-CnIA for
the muscular vs. neuronal (alpha7) nicotinic receptors was 2 orders of
magnitude (IC50 of 0.19 uM for competition with [ 125 I]-alpha-Bgtx binding
to muscular nicotinic receptors in Torpedo vs. 14.8 uM for
neuronal alpha7 nicotinic receptors expressed in HEK cells). The
80-fold difference in the binding affinity between the muscular and neuronal
alpha7 receptor is in accordance with the lack of effects previously observed
on ganglionic preparations with alpha-MI. The specificity for muscular
versus neuronal nAChRs of alpha-CnIA was studied in bovine chromaffin
cells (neuronal-type nAChR) where the synthetic alpha-CnIA (1 uM) was
unable to block the nicotine-evoked calcium influx in these cells (P. Favreau,
C. Mattei, and J. Molgo, unpublished data). This can be compared to the
potent inhibition of nicotine-induced catecholamine release from bovine
chromaffin cells by alpha-EpI from Conus episcopatus (Loughnan et al 1998).
Thus, alpha-CnIA appears to be a potent and
blocker of the muscular subtype nAChRs as evidenced by electro-physiological
recordings with amphibian and mammalian isolated neuromuscular preparations.