Abstract
We have isolated a 16-amino acid peptide from the venom of the marine snail Conus magus which potently blocks nicotinic acetylcholine receptors (nAChRs) composed of alpha3beta2 subunits. This peptide, named alpha-conotoxin MII, was identified by electrophysiologically screening venom fractions against cloned nicotinic receptors expressed in Xenopus oocytes. The peptide's structure, which has been confirmed by mass spectrometry and total chemical synthesis, differs significantly from those of all previously isolated alpha-conotoxins. Disulfide bridging, however, is conserved. The toxin blocks the response to acetylcholine in oocytes expressing alpha3beta2 nAChRs with an IC50 of 0.5 nM and is 2-4 orders of magnitude less potent on other nAChR subunit combinations. We have recently reported the isolation and characterization of alpha-conotoxin ImI, which selectively targets homomeric alpha7 neuronal nAChRs. Yet other alpha-conotoxins selectively block the muscle subtype of nAChR. Thus, it is increasingly apparent that alpha-conotoxins represent a significant resource for ligands with which to probe structure-function relationships of various nAChR subtypes.
Highlights
The muscle subtype of nicotinic acetylcholine receptor1 is one of the best understood ligand-gated channels due in part to the availability of a large number of protein and small molecule ligands which serve as specific probes for this channel
Purification and Characterization of ␣-Conotoxin MII—Serial dilutions of a 50 mg/ml ND96 buffer extract of crude C. magus venom were tested for their ability to block the AChinduced current in Xenopus oocytes expressing ␣32 nicotinic acetylcholine receptors (nAChRs)
We examined the effect of the toxin on other nAChR subunit combinations expressed in oocytes
Summary
The muscle subtype of nicotinic acetylcholine receptor (nAChR) is one of the best understood ligand-gated channels due in part to the availability of a large number of protein and small molecule ligands which serve as specific probes for this channel. A number of valuable nicotinic antagonists have been described, few are highly subtype-selective, in the case of neuronal nAChRs. d-Tubocurarine, an alkaloid from the Chondrodendron tomentosum bush, used for centuries as an arrow poison to kill wild game, blocks both muscle and neuronal nAChRs [3] In addition it binds to all neuronal nicotinic receptors with more or less similar affinities [4]. Lophotoxin, a small cyclic diterpene, is used by the soft shell coral Lophogorgia rigida to discourage its consumption by fish [5] This toxin forms a covalent bond with Tyr190 of the ␣-subunit of Torpedo nAChRs, irreversibly blocking the binding of ACh to the receptor [6, 7]. In contrast to snake ␣-toxins (ϳ60 – 80 amino acids), ␣-conotoxins are much smaller (ϳ12–25 amino acids), a feature which has allowed them to be readily chemically synthesized [13]. ␣-Conotoxins, which target the
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