Identification of all residues involved in the recognition and binding of cholinergic ligands (e.g. agonists, competitive antagonists, and noncompetitive agonists) is a primary objective to understand which structural components are related to the physiological function of the nicotinic acetylcholine receptor (AChR). The picture for the localization of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are located mainly on both alpha subunits in a pocket approximately 30-35 Angstrom above the surface membrane. Since both alpha subunits are identical, the observed high and low affinity for different ligands on the receptor is conditioned by the interaction of the alpha subunit with other non-alpha subunits. This molecular interaction takes place at the interface formed by the different subunits. For example, the high affinity acetylcholine (ACh) binding site of the muscle-type AChR is located on the alpha delta subunit interface, whereas the low-affinity ACh binding site is located on the alpha gamma subunit interface. Regarding homomeric AChRs (e.g. alpha 7, alpha 8, and alpha 9), up to five binding sires may be located on the alpha alpha subunit interfaces. From the point of view of subunit arrangement, the gamma subunit is in between both alpha subunits and the delta subunit follows the alpha aligned in a clockwise manner from the gamma. Although some competitive antagonists such as lophotoxin and alpha-bungarotoxin bind to the same high- and low-affinity sites as ACh, other cholinergic drugs may bind with opposite specificity. For instance, the location of the high- and the low-affinity binding site for curare-related drugs as well as for agonists such as the alkaloid nicotine and the potent analgesic epibatidine (only when the AChR is in the desensitized state) is determined by the alpha gamma and the alpha delta subunit interface, respectively. The case of alpha-conotoxins (alpha-CoTxs) is unique since each alpha-CoTx from different species is recognized by a specific AChR type. In addition, the specificity of alpha-CoTxs for each subunit interface is species-dependent. In general terms we may state that both a subunits carry the principal component for the agonist/competitive antagonist binding sites, whereas the non-alpha subunits bear the complementary component. Concerning homomeric AChRs, both the principal and the complementary component exist on the alpha subunit. The principal component on the muscle-type AChR involves three loops-forming binding domains (loops A-C). Loop A (from mouse sequence) is mainly formed by residue Y-93, loop B is molded by amino acids W-149, Y-152, and probably G(153), while loop C is shaped by residues Y-190, C-192, C-193, and Y-198 The complementary component corresponding to each non-alpha subunit probably contributes with at least four loops. More specifically, the loops at the gamma subunit are: loop D which is formed by residue K-34, loop E that is designed by W-55 and E-57, loop F which is built by a stretch of amino acids comprising L-109, S-111, C-115, l(116), and Y-117, and finally loop G that is shaped by F-172 and by the negatively-charged amino acids D-174 and E-183. The complementary component on the delta subunit, which corresponds to the high-affinity ACh binding site, is formed by homologous loops. Regarding alpha-neurotoxins, several snake and alpha-CoTxs bear specific residues that are energetically coupled with their corresponding pairs on the AChR binding site. The principal component for snake alpha-neurotoxins is located on the residue sequence alpha 1W(184)-D-200 which includes loop C. Tn addition, amino acid sequence 55-74 from the alpha 1 subunit (which includes loop E), and residues gamma L-119 (close to loop F) and gamma E-176 (close to loop G) at the low-affinity binding site, or delta L-121 (close to the homologous region of loop G) at the high-affinity binding site, are involved in snake alpha-neurotoxin binding. The above expounded evidence indicates that each cholinergic molecule binds to specific residues which form overlapping binding sites on the AChR. Monoclonal antibodies have been of fundamental importance in the elucidation of several aspects of the biology of the AChR. Interestingly, certain antibodies partially overlap with the agonist/competitive antagonist binding sites at multiple points of contact. In this regard, a monoclonal antibody directed against the high-affinity ACh binding site (alpha delta subunit interface) induced a structural change on the AChR where the low-affinity ACh locus (alpha gamma subunit interface) approached to the lipid membrane. The alpha subunits also carry the binding site for noncompetitive agonists. Noncompetitive agonists such as the acetylcholinesterase inhibitor (-)-physostigmine, the alkaloid galanthamine, and the opioid derivative codeine are molecules that weakly activate the receptor without interacting with the classical agonist binding sites. This binding site was found to be located at K-125 in an amphipathic domain of the extracellular portion of the alpha 1 subunit. interestingly, the neurotransmitter 5-hydroxytryptamine (5-HT) also binds to this site and enhances the agonist-induced ion flux activity. This suggests that 5-HT may act as an endogenous modulator (probably as co-agonist) of neuronal-type AChRs. The enhancement of the agonist-evoked currents elicited by noncompetitive agonists seems to be physiologically more important than their weak agonist properties. (C) 2000 Elsevier Science Ltd. All rights reserved.
