G. Edward Cartier

α-Conotoxin AuIB Selectively Blocks α3β4 Nicotinic Acetylcholine Receptors and Nicotine-Evoked Norepinephrine Release

Neuronal nicotinic acetylcholine receptors (nAChRs) with putative α3β4-subunits have been implicated in the mediation of signaling in various systems, including ganglionic transmission peripherally and nicotine-evoked neurotransmitter release centrally. However, progress in the characterization of these receptors has been hampered by a lack of α3β4-selective ligands. In this report, we describe the purification and characterization of an α3β4 nAChR antagonist, α-conotoxin AuIB, from the venom of the “court cone,”Conus aulicus. We also describe the total chemical synthesis of this and two related peptides that were also isolated from the venom. α-Conotoxin AuIB blocks α3β4 nAChRs expressed in Xenopus oocytes with an IC50 of 0.75 μm, a kon of 1.4 × 106 min-1m−1, a koffof 0.48 min-1, and aKd of 0.5 μm. Furthermore, α-conotoxin AuIB blocks the α3β4 receptor with >100-fold higher potency than other receptor subunit combinations, including α2β2, α2β4, α3β2, α4β2, α4β4, and α1β1γδ. Thus, AuIB is a novel, selective probe for α3β4 nAChRs. AuIB (1–5 μm) blocks 20–35% of the nicotine-stimulated norepinephrine release from rat hippocampal synaptosomes, whereas nicotine-evoked dopamine release from striatal synaptosomes is not affected. Conversely, the α3β2-specific α-conotoxin MII (100 nm) blocks 33% of striatal dopamine release but not hippocampal norepinephrine release. This suggests that in the respective systems, α3β4-containing nAChRs mediate norepinephrine release, whereas α3β2-containing receptors mediate dopamine release.

Speciation of Cone Snails and Interspecific Hyperdivergence of Their Venom Peptides: Potential Evolutionary Significance of Intronsa

ABSTRACT: All 500 species of cone snails (Conus) are venomous predators. From a biochemical/genetic perspective, differences among Conus species may be based on the 50‐200 different peptides in the venom of each species. Venom is used for prey capture as well as for interactions with predators and competitors. The venom of every species has its own distinct complement of peptides. Some of the interspecific divergence observed in venom peptides can be explained by differential expression of venom peptide superfamilies in different species and of peptide superfamily branching in various Conus lineages into pharmacologic groups with different targeting specificity. However, the striking interspecific divergence of peptide sequences is the dominant factor in the differences observed between venoms. The small venom peptides (typically 10‐35 amino acids in length) are processed from larger prepropeptide precursors (ca. 100 amino acids). If interspecific comparisons are made between homologous prepropeptides, the three different regions of a Conus peptide precursor (signal sequence, pro‐region, mature peptide) are found to have diverged at remarkably different rates. Analysis of synonymous and nonsynonymous substitution rates for the different segments of a prepropeptide suggests that mutation frequency varies by over an order of magnitude across the segments, with the mature toxin region undergoing the highest rate. The three sections of the prepropeptide which exhibit apparently different mutation rates are separated by introns. This striking segment‐specific rate of divergence of Conus prepropeptides suggests a role for introns in evolution: exons separated by introns have the potential to evolve very different mutation rates. Plausible mechanisms that could underlie differing mutational frequency in the different exons of a gene are discussed.

Differential targeting of nicotinic acetylcholine receptors by novel alphaA-conotoxins.

We describe the isolation and characterization of two peptide toxins from Conus ermineus venom targeted to nicotinic acetylcholine receptors (nAChRs). The peptide structures have been confirmed by mass spectrometry and chemical synthesis. In contrast to the 12–18 residue, 4 Cys-containing α-conotoxins, the new toxins have 30 residues and 6 Cys residues. The toxins, named αA-conotoxins EIVA and EIVB, block both Torpedo and mouse α1-containing muscle subtype nAChRs expressed in Xenopus oocytes at low nanomolar concentrations. In contrast to α-bungarotoxin, αA-EIVA is inactive at α7-containing nAChRs even at micromolar concentrations. In this regard, αA-EIVA is similar to the previously described α-conotoxins (e.g. α-MI and α-GI) which also selectively target α1- versus α7-containing nAChRs. However, α-MI and α-GI discriminate between the α/δversus α/γ subunit interfaces of the mouse muscle nAChR with 10,000-fold selectivity. In contrast, αA-conotoxin EIVA blocks both the α/γ site and α/δ site with equally high affinity but with distinct kinetics. The αA-conotoxins thus represent novel probes for the α/γ as well as the α/δ binding sites of the nAChR.

Composition and therapeutic utility of conotoxins from genus Conus. Patent status 1996 - 2000

With an exponentially increasing body of scientific evidence pointing toward the potential of conotoxins for treatment of a wide variety of nervous system and associated neurological disorders, there has been an explosion of activity in this patent area with more than eighty new patents and PCT publications in the past five years. With the emergence of ziconotide (SNX-111, ω-conotoxin MVIIA) as the first clinically used conotoxin for treatment of a neurological disorder, the first part of the new millennium is likely to see many more new filings in this field. The majority of the applications from this period focus on those classes of conopeptides that interact with nicotinic acetylcholine receptors (nAChRs) together with those that block voltage-gated ion channels. This arena has to date been dominated by three research groups: Neurex (a wholly-owned subsidiary of Elan, South San Francisco, CA, USA), Xenome and the Institute for Molecular Bioscience (IMB), University of Queensland (Melbourne, Australia) and Cognetix (Salt Lake City, UT, USA) together with the University of Utah Research Foundation and the Salk Institute for Biological Studies (La Jolla, CA, USA).