Nicotinic acetylcholine receptors (nAChRs) are integral membrane proteins and prototypic members of the ligand-gated ion-channel superfamily, which has precursors in the prokaryotic world. They are formed by the assembly of five transmembrane subunits, selected from a pool of 17 homologous polypeptides (a1-10, b1-4, c, d, and e). There are many nAChR subtypes, each consisting of a specific combination of subunits, which mediate diverse physiological functions. They are widely expressed in the central nervous system, while, in the periphery, they mediate synaptic transmission at the neuromuscular junction and ganglia. nAChRs are also found in non-neuronal ⁄ nonmuscle cells (keratinocytes, epithelia, macrophages, etc.). Extensive research has determined the specific function of several nAChR subtypes. nAChRs are now important therapeutic targets for various diseases, including myasthenia gravis, Alzheimer's and Parkinson's diseases, and schizophrenia, as well as for the cessation of smoking. However, knowledge is still incomplete, largely because of a lack of high-resolution X-ray structures for these molecules. Nevertheless, electron microscopy studies on 2D crystals of nAChR from fish electric organs and the determination of the high-resolution X-ray structure of the acetylcholine binding protein (AChBP) from snails, a homolog of the extracellular domain of the nAChR, have been major steps forward and the data obtained have important implications for the design of subtype-specific drugs. Here, we review some of the latest advances in our understanding of nAChRs and their involvement in physiology and pathology.

The N-terminal extracellular domain (ECD; amino acids 1-208) of the neuronal nicotinic acetylcholine receptor (AChR) ␣7 subunit, the only human AChR subunit known to assemble as a homopentamer, was expressed as a glycosylated form in the yeast Pichia pastoris in order to obtain a native-like model of the extracellular part of an intact pentameric nicotinic AChR. This molecule, ␣7-ECD, although able to bind the specific ligand ␣-bungarotoxin, existed mainly in the form of microaggregates. Substitution of Cys-116 in the ␣7-ECD with serine led to a decrease in microaggregate size. A second mutant form, ␣7-ECD(C116S,Cys-loop), was generated in which, in addition to the C116S mutation, the hydropho- ؊7 M, K i ‫؍‬ 1 ؋ 10 ؊5 M, and K i ‫؍‬ 0.9 ؋ 10 ؊2 M, respectively). All three constructs were expressed as glycosylated forms, but in vitro deglycosylation reduced the heterogeneity without affecting their ligand binding properties. These results show that ␣7-ECD(C116S,Cys-loop) was expressed in P. pastoris as an oligomer (probably a pentamer) with a near native conformation and that its deglycosylated form seems to be suitable starting material for structural studies on the ligand-binding domain of a neurotransmitter receptor.

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Photoaffinity labeling is a powerful tool for the characterization of the molecular basis of ligand binding to acceptor molecules, which provides important insights for mapping the bimolecular interfaces. The autoimmune disease myasthenia gravis is caused by autoantibodies against the acetylcholine receptor (AChR). The majority of the anti‐AChR antibodies bind to the “main immunogenic region” (MIR) of the AChR. To identify the contact points between the complementarity determining regions of the anti‐MIR antibodies that recognize the MIR contact sites of the AChR, we present here three photoreactive dodecapeptide MIR analogues containing the photolabel p‐benzoyl‐L‐phenylalanine (Bpa) moiety, either in position 1 or 11. The structure of the produced 12‐mers was analyzed using two‐dimensional 1H‐NMR spectroscopy, whereas their binding to anti‐MIR monoclonal antibodies (mAbs) was determined by immunochemical assays. In all cases the modifications resulted in conservation of the β‐turn conformation of the N‐terminus, which has been proved essential for antibody recognition and increased anti‐MIR binding relative to the MIR decapeptide. © 2001 John Wiley & Sons, Inc. Biopolymers 56: 37–46, 2001