Conotoxins (Ctx) form a large family of peptide toxins from cone snail venoms that act on a broad spectrum of ion channels and receptors. The subgroup alpha-Ctx specifically and selectively binds to subtypes of nicotinic acetylcholine receptors (nAChRs), which are targets for treatment of several neurological disorders. Here we present the structure at a resolution of 2.4 A of alpha-Ctx PnIA (A10L D14K), a potent blocker of the alpha(7)-nAChR, bound with high affinity to acetylcholine binding protein (AChBP), the prototype for the ligand-binding domains of the nAChR superfamily. Alpha-Ctx is buried deep within the ligand-binding site and interacts with residues on both faces of adjacent subunits. The toxin itself does not change conformation, but displaces the C loop of AChBP and induces a rigid-body subunit movement. Knowledge of these contacts could facilitate the rational design of drug leads using the Ctx framework and may lead to compounds with increased receptor subtype selectivity.

Iodinated [125I] weak toxin from Naja kaouthia (WTX) cobra venom was injected into mice, and organ‐specific binding was monitored. Relatively high levels of [125I]WTX were detected in the adrenal glands. Rat adrenal membranes were therefore used for analysis of [125I]WTX‐binding sites. Specific [125I]WTX binding was partially inhibited by both α‐cobratoxin, a blocker of the α7 and muscle‐type nicotinic acetylcholine receptors (nAChRs), and by atropine, an antagonist of the muscarinic acetylcholine receptor (mAChR). Binding to rat adrenal nAChR had a Kd of 2.0 ± 0.8 μm and was inhibited by α‐cobratoxin but not by a short‐chain α‐neurotoxin antagonist of the muscle‐type nAChR, suggesting a specific interaction with the α7‐type nAChR. WTX binding was reduced not only by atropine but also by other muscarinic agents (oxotremorine and muscarinic toxins from Dendroaspis angusticeps), indicating an interaction with mAChR. This interaction was further characterized using individual subtypes of human mAChRs expressed in Chinese hamster ovary cells. WTX concentrations up to 30 μm did not inhibit binding of [3H]acetylcholine to any subtype of mAChR by more than 50%. Depending on receptor subtype, WTX either increased or had no effect on the binding of the muscarinic antagonist [3H]N‐methylscopolamine, which binds to the orthosteric site, a finding indicative of an allosteric interaction. Furthermore, WTX alone activated G‐protein coupling with all mAChR subtypes and reduced the efficacy of acetylcholine in activating G‐proteins with the M1, M4, and M5 subtypes. Our data demonstrate an orthosteric WTX interaction with nAChR and an allosteric interaction with mAChRs.

Different snake venom neurotoxins block distinct subtypes of nicotinic acetylcholine receptors (nAChR). Short-chain ␣-neurotoxins preferentially inhibit muscle-type nAChRs, whereas longchain ␣-neurotoxins block both muscle-type and ␣7 homooligomeric neuronal nAChRs. An additional disulfide in the central loop of ␣-and -neurotoxins is essential for their action on the ␣7 and ␣3␤2 nAChRs, respectively. Design of novel toxins may help to better understand their subtype specificity. To address this problem, two chimeric toxins were produced by bacterial expression, a short-chain neurotoxin II Naja oxiana with the grafted disulfidecontaining loop from long-chain neurotoxin I from N. oxiana, while a second chimera contained an additional A29K mutation, the most pronounced difference in the central loop tip between long-chain ␣-neurotoxins and -neurotoxins. The correct folding and structural stability for both chimeras were shown by 1 H and 1 H-15 N NMR spectroscopy. Electrophysiology experiments on the nAChRs expressed in Xenopus oocytes revealed that the first chimera and neurotoxin I block ␣7 nAChRs with similar potency (IC 50 6.1 and 34 nM, respectively). Therefore, the disulfide-confined loop endows neurotoxin II with full activity of long-chain ␣-neurotoxin and the C-terminal tail in neurotoxin I is not essential for binding. The A29K mutation of the chimera considerably diminished the affinity for ␣7 nAChR (IC 50 126 nM) but did not convey activity at ␣3␤2 nAChRs. Docking of both chimeras to ␣7 and ␣3␤2 nAChRs was possible, but complexes with the latter were not stable at molecular dynamics simulations. Apparently, some other residues and dimeric organization of -neurotoxins underlie their selectivity for ␣3␤2 nAChRs. Nicotinic acetylcholine receptors (nAChRs)2 belong to the class of ligand-gated ion channels (1-3). All nAChRs are composed of five homologous subunits, which span the plasma membrane to form a transmembrane ion-conducting pore. The muscle-type receptors are composed of four types of subunits, ␣2␤␥␦ in the fetal and ␣2␤⑀␦ in the mature receptor. The best studied nAChR of this type is the receptor from electric organ of ray Torpedo marmorata (4). Neuronal receptors consist of two types of subunits, ␣ (␣2-␣10) and ␤ (␤2-␤4), and can have homooligomeric (for example, ␣7-type nAChR) or heterooligomeric (for example, ␣3␤2-type receptor) composition. Neuronal nAChRs are widely expressed both pre-and postsynaptically, and presynaptic receptors modulate synaptic activity and the release of different neurotransmitters. Neuronal nAChRs are found also in non-neuronal tissues, such as keratinocytes and pulmonary and other tissues (5-8).Because of their role in cellular communication, nAChRs play important roles in the normal functioning of the organism. Dysfunctions of these receptors have been proposed to contribute to a number of disorders of the central and peripheral nervous systems such as schizophrenia, epilepsy, depression, nicotine and alcohol dependence, Alzheimer disease, Parkinson disease, and autis...

a-Conotoxins are a group of relatively short peptides (12-19 amino acid residues, two disulfide bridges) from the venom of poisonous marine snails of the Conus

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