The T-superfamily of Conotoxins

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Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. ␣ Subunits, together with ␤2 and/or ␤4 subunits, form ligand-binding sites at ␣/␤ subunit interfaces. Predatory marine snails of the genus Conus are a rich source of nAChR-targeted peptides. Using conserved features of the ␣-conotoxin signal sequence and 3 -untranslated sequence region, we have cloned a novel gene from the fisheating snail, Conus bullatus; the gene codes for a previously unreported ␣-conotoxin with unusual 4/4 spacing of amino acids in the two disulfide loops. Chemical synthesis of the predicted mature toxin was performed. The resulting peptide, ␣-conotoxin BuIA, was tested on cloned nAChRs expressed in Xenopus oocytes. The peptide potently blocks numerous rat nAChR subtypes, with highest potency for ␣3-and chimeric ␣6-containing nAChRs; BuIA blocks ␣6/␣3␤2 nAChRs with a 40,000-fold lower IC 50 than ␣4␤2 nAChRs. The kinetics of toxin unblock are dependent on the ␤ subunit. nAChRs with a ␤4 subunit have very slow off-times, compared with the corresponding ␤2 subunit-containing nAChR. In each instance, rat ␣x␤4 may be distinguished from rat ␣x␤2 by the large difference in time to recover from toxin block. Similar results are obtained when comparing mouse ␣3␤2 to mouse ␣3␤4, and human ␣3␤2 to human ␣3␤4, indicating that the ␤ subunit dependence extends across species. Thus, ␣-conotoxin BuIA also represents a novel probe for distinguishing between ␤2-and ␤4-containing nAChRs.

Section: Methodsmentioning

confidence: 99%

α-Conotoxin BuIA, a Novel Peptide from Conus bullatus, Distinguishes among Neuronal Nicotinic Acetylcholine Receptors

Azam

Dowell

Watkins

et al. 2005

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117

“…The peptide was removed from the resin and precipitated. A two-step oxidation protocol was used to -fold selectively the peptides as described previously (14). Briefly, the disulfide bridge between Cys 2 and Cys 8 was closed by dripping the peptide into an equal volume of 20 mM potassium ferricyanide, 0.1 M Tris-HCl, pH 7.5.…”

Section: Methodsmentioning

confidence: 99%

α-Conotoxin OmIA Is a Potent Ligand for the Acetylcholine-binding Protein as Well as α3β2 and α7 Nicotinic Acetylcholine Receptors

Talley

Olivera

Han

et al. 2006

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The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the ␣-subunit of nicotinic acetylcholine receptors and in particular the homomeric ␣7 nicotinic receptor. We report the isolation and characterization of an ␣-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the ␣7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the ␣3␤2 nAChR indicating that ␣-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of ␣3␤2 nAChRs. ␣-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17-amino-acid, two-disulfide bridge peptide. The ligand is the first ␣-conotoxin with higher affinity for the closely related receptor subtypes, ␣3␤2 versus ␣6␤2, and selectively blocks these two subtypes when compared with ␣2␤2, ␣4␤2, and ␣1␤1␦⑀ nAChRs. Nicotinic acetylcholine receptors (nAChRs)2 are found in the neuromuscular junction, peripheral nervous and central nervous systems of both invertebrates and vertebrates. These receptors play essential roles in mediating synaptic transmission and modulating the release of a variety of neurotransmitters. Different molecular forms of the nAChR are comprised of homopentameric (␣7 and ␣9) and heteropentameric (e.g. ␣3␤2, ␣4␤2, and ␣1␤1␥⑀) arrangements of subunits that have discrete anatomical locations and distinct physiological functions. Dysfunction or dysregulation of nAChRs is implicated in a variety of neuropsychiatric disease states including schizophrenia, Parkinson, Alzheimer, depression and nicotine addiction (1). Several drug discovery programs aim to develop specific drugs that selectively act on subtypes of nAChRs.The AChBP, synthesized in molluskan glial cells, is proposed to function as a modulator of synaptic ACh transmission. ACh, acting on a glial nAChR, induces cellular release of AChBP. AChBP, in turn, binds presynaptically released ACh, acting as a synaptic buffer to dampen synaptic transmission (2). AChBP has sequence similarity (15-28% identity) with subunits of the cysteine loop ligand-gated ion channel family that includes nAChRs, GABA A , GABA C , 5-hydroxytryptophan type 3, and glycine receptors (3). These receptors assemble as heteromeric or homomeric pentamers of subunits, and each subunit has an NH 2 -terminal extracellular ligand-binding domain and four COOH-terminal transmembrane spans that serve as the channel pore-forming domain. AChBP is homologous to the extracellular domain of the cysteine-loop family. The crystal structure of AChBP has provided a structural template for examining ligand recognition in nAChRs by spectroscopic analysis of conjugated fluorophores, structural modeling, dynamics of deuterium/hydrogen exchange, computational docking of ligands, and direct monitoring of ligand occupancy by changes in intrinsic Trp fluorescence (4 -8).Despite several conserved ...

“…Amino acid derivatives were from Bachem (Torrance, CA). The peptides were removed from the resin and precipitated; a two-step oxidation protocol was used to selectively fold the peptides as described previously (14). Briefly, the first disulfide bridge was closed by dripping the peptide into an equal volume of 20 mM potassium ferricyanide, 0.1 M Tris, pH 7.5.…”

Section: Methodsmentioning

confidence: 99%

α-Conotoxin GIC from Conus geographus, a Novel Peptide Antagonist of Nicotinic Acetylcholine Receptors

McIntosh

Dowell

Watkins

et al. 2002

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107

Many venomous organisms produce toxins that disrupt neuromuscular communication to paralyze their prey. One common class of such toxins comprises nicotinic acetylcholine receptor antagonists (nAChRs). Thus, most toxins that act on nAChRs are targeted to the neuromuscular subtype. The toxin characterized in this report, ␣-conotoxin GIC, is a most striking exception. The 16-amino acid peptide was identified from a genomic DNA clone from Conus geographus. The predicted mature toxin was synthesized, and synthetic toxin was used in all studies described. ␣-Conotoxin GIC shows no paralytic activity in fish or mice. Furthermore, even at concentrations up to 100 M, the peptide has no detectable effect on the human muscle nicotinic receptor subtype heterologously expressed in Xenopus oocytes. In contrast, the toxin has high affinity (IC 50 Ϸ1.1 nM) for the human ␣3␤2 subunit combination, making it the most neuronally selective nicotinic antagonist characterized thus far. Although ␣-conotoxin GIC shares some sequence similarity with ␣-conotoxin MII, which is also a potent ␣3␤2 nicotinic antagonist, it is much less hydrophobic, and the kinetics of channel block are substantially different. It is noteworthy that the nicotinic ligands in C. geographus venom fit an emerging pattern in venomous predators, with one nicotinic antagonist targeted to the muscle subtype (thereby causing paralysis) and a second nicotinic antagonist targeted to the ␣3␤2 nAChR subtype (possibly inhibiting the fight-orflight response).