The nicotinic acetylcholine receptor (

The alpha-conotoxins are paralytic peptide toxins from Indo-Pacific cone snails. This paper presents a detailed analysis of how alpha-conotoxins inhibit [125I]-alpha-bungarotoxin (125I-BTX) equilibrium binding to the acetylcholine receptor (AChR) from electric organ of Torpedo californica and Torpedo nobiliana. All three alpha-conotoxins studied, SI, GI, and MI, completely inhibited 125I-BTX binding with the same order of potency in both species (MI approximately GI > SI approximately d-tubocurarine). BTX-concentration curves showed that this inhibition is competitive. However, while SI appeared to bind to a homogeneous population of sites, both GI and MI displayed curare-like heterogeneous binding. Studies using partially-blocked AChR demonstrated that both GI and MI display different affinities toward the two agonist sites, much like small curariform antagonists do. The high-affinity site for these two alpha-conotoxins is also the high-affinity d-tubocurarine site, which is believed to be located at the alpha gamma-subunit interface. The high-affinity binding of MI and GI was of the same order of magnitude as that of d-tubocurarine; however, their affinity for the other agonist site was somewhat greater than that of dTC, resulting in less site selectivity. Despite being homologous to GI and MI, SI did not distinguish between the two sites. A possible molecular basis for this difference is presented.

Cembranoids are cyclic diterpenoids found in tobacco and in marine invertebrates. The present study established that tobacco cembranoids inhibit behavioral sensitization to nicotine in rats and block several types of nicotine acetylcholine receptors (AChRs). 1) At the behavioral level, rat locomotor activity induced by nicotine was significantly increased after seven daily nicotine injections. This sensitization to nicotine was blocked by mecamylamine (1 mg/kg) and by the cembranoids eunicin, eupalmerin acetate (EUAC), and (4R)-2,7,11-cembratriene-4-6-diol (4R), each at 6 mg/kg. None of these compounds modified locomotor activity of nonsensitized rats. 2) In cells expressing human AChRs, cembranoids blocked carbamoylcholine-induced (86)Rb(+) flux with IC(50) in the low micromolar range. The cell lines used were the SH-EP1-halpha4beta2 cell line heterologously expressing human alpha4beta2-AChR, the SH-SY5Y neuroblastoma line naturally expressing human ganglionic alpha3beta4-AChR, and the TE671/RD cell line naturally expressing embryonic muscle alpha1beta1gammadelta-AChR. The tobacco cembranoids tested were 4R and its diastereoisomer 4S, and marine cembranoids tested were EUAC and 12,13-bisepieupalmerin. 3) At the molecular level, tobacco (4R and 4S) and marine (EUAC) cembranoids blocked binding of the noncompetitive inhibitor [(3)H]tenocyclidine to AChR from Torpedo californica electric organ. IC(50) values were in the submicromolar to low-micromolar range, with 4R displaying an order of magnitude higher potency than its diastereoisomer, 4S.