Conus peptides: novel probes for nicotinic acetylcholine receptor structure and function

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Abstract

Conus is a genus of predatory marine snails that uses venom to capture prey. Among the neurotoxins widely utilized by the cone snails are the α-conotoxins which are disulfide-rich peptides that target muscle or neuronal subtypes of nicotinic acetylcholine receptors. The small size and receptor subtype specificity of these peptides make them particularly useful for characterizing both native and heterologously expressed nicotinic receptors. In this report, we demonstrate that α-conotoxin MII potently blocks β3-containing neuronal nicotinic receptors. Furthermore, initial evidence suggests that subpopulations of α3β2β3-containing receptors are differentially sensitive to α-conotoxin MII. Thus, α-conotoxin MII promises to be a useful tool for studying neuronal nicotinic receptors containing the β3 subunit.

Introduction

Plants and animals historically have been a rich source of antagonists of nicotinic acetylcholine receptors. One of the more recently explored sources is the marine snail Conus, a venomous predator. There are over 500 species of cone snails; each venom thus far examined contains multiple compounds targeted to neuronal nicotinic receptors. Many of these compounds are disulfide-rich peptides. Their small size has made them amenable to chemical synthesis (see McIntosh et al., 1999 for review).

The initial α-conotoxins characterized (e.g., α-conotoxin GI and α-conotoxin MI) potently block muscle but not neuronal nicotinic receptors Luetje et al., 1990, Johnson et al., 1995. With regard to muscle nicotinic receptors, in mammalian muscle, these α-conotoxins show five orders-of-magnitude selectivity for the α/δ vs. the α/γ subunit interface (Sine et al., 1995). More recently, α-conotoxins that target neuronal nicotinic receptors have been characterized. α-Conotoxin ImI was isolated from a worm-hunting cone; ImI selectively blocks the mammalian α7 receptor (Johnson et al., 1995). The α7 subtype of receptor is notable for its rapid desensitization. It is of interest that α-conotoxin ImI also blocks apparently rapidly desensitizing receptors populations in invertebrates. In Aplysia, α-conotoxin ImI selectively blocks the rapidly desensitizing portion of the acetylcholine-gated chloride current (Kehoe and McIntosh, 1998). In locusts, α-conotoxin ImI blocks the rapidly desensitizing portion of the acetylcholine-induced current in thoracic ganglion (Van den Beukel et al., 1998). α-Conotoxin MII, isolated from a fish-hunting cone, selectively blocks the rat α3β2 receptor with subnanomolar affinity (Cartier et al., 1996). In contrast to α-conotoxin ImI, which blocks rapidly desensitizing currents, α-conotoxin MII selectively blocks the slowly decaying current in chick ciliary ganglion (Ullian et al., 1997). In frog sympathetic ganglia, α-conotoxin MII and α-conotoxin ImI differentially block synapses on B vs. C neurons (Tavazoie et al., 1997). In addition to fish- and worm-hunting cone snails, α-conotoxins have also been isolated from mollusc-hunting snails. One example is α-conotoxin AuIB which selectively targets rat α3β4 neuronal receptors (Luo et al., 1998) and putative α3β4-containing neuronal nicotinic receptors present in rat habenula neurons (Quick et al., 1999). α-Conotoxins MII and AuIB have been used to selectively block nicotine-induced dopamine release and nicotine-evoked norepinephrine release, respectively Kulak et al., 1997, Kaiser et al., 1998, Luo et al., 1998.

In this study we report that, in addition to α3β2 receptors, α-conotoxin MII potently blocks α3β2β3 receptors expressed in Xenopus oocytes. Initial results also suggest that there are distinct populations of α3β2β3 receptors that are differentially sensitive to α-conotoxin MII.

Section snippets

Materials

α-Conotoxins were synthesized as previously described (Cartier et al., 1996). Toxins were stored in lyophilized form at −20° C until use.

Cloning of neuronal nicotinic receptor subunits

Clones of the human nicotinic acetylcholine receptor subunits were generated by reverse transcription–polymerase chain reaction (PCR). Sequences in the 5′ and 3′ untranslated regions of each of the neuronal nicotinic receptor mRNAs were used to design PCR primers to amplify the complete open reading frame of each subunit. Restriction enzyme sites were

Results

Oocytes injected with cRNA for the β3 subunit alone, the α3 subunit alone, the β2 subunit alone, the β2 and β3 subunits in combination, and the α3 and β3 subunits in combination, all failed to express active receptor as tested with application of 300 μM acetylcholine. In contrast, oocytes injected with either α3β2 or α3, β2 and β3 cRNA reliably expressed active receptor. In oocytes expressing α3β2 receptors, acetylcholine elicited a relatively symmetrical-shaped response (Fig. 1A). In oocytes

Discussion

α-Conotoxins from different species of cone snails have been used to selectively antagonize neuronal nicotinic receptors with different subunits (see Table 1). It should be noted that the effectiveness of these toxins for a given subtype of nicotinic receptor may vary according to the species of the organism producing the receptor.

In this report, we have shown that the human β3 subunit combines with α3 and β2 subunits to form functional receptors. In addition, we have shown that α-conotoxin MII

Acknowledgements

This work was supported by NIH grants MH53631 and GM48677.

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