Models of the extracellular ligand-binding domain of nicotinic acetylcholine receptors (nAChRs), which are pentameric integral membrane proteins, are attractive for structural studies because they potentially are water-soluble and better candidates for x-ray crystallography and because their smaller size is more amenable for NMR spectroscopy. The complete N-terminal extracellular domain is a promising foundation for such models, based on previous studies of ␣7 and muscle-type subunits. Specific design requirements leading to high structural fidelity between extracellular domain nAChRs and full-length nAChRs, however, are not well understood. To study these requirements in heteromeric nAChRs, the extracellular domains of ␣4 and ␤2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with ␣4␤2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain ␣4␤2 nAChRs formed from subunits with M1 were nearly identical to affinities of ␣4␤2 nAChRs when measured with [ 3 H]epibatidine, cytisine, nicotine, and acetylcholine. Velocity sedimentation suggested that these extracellular domain nAChRs predominantly formed pentamers. The yield of these extracellular domain nAChRs was about half the yield of ␣4␤2 nAChRs. In contrast, [3 H]epibatidine binding was not detected from the extracellular domain ␣4 and ␤2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both ␣4 and ␤2 play an important role for efficient expression of extracellular domain ␣4␤2 nAChRs that are high fidelity structural models of full-length ␣4␤2 nAChRs. Nicotinic acetylcholine receptors (nAChRs)2 are ligand-gated ion channels expressed mainly in the nervous system and at the neuromuscular junction (1-3), although they also are expressed elsewhere (4). They are members of the superfamily of nicotinoid receptors, which includes ␥-aminobutyric acid (GABA) type A and C, glycine, and serotonin 5-HT3 receptors. They contain five homologous subunits, with subunits designated ␣1-␣10, ␤1-␤4, ␦, ␥, and ⑀. Each subunit is an integral membrane protein with a large, ligand-binding N-terminal extracellular domain, four transmembrane domains (M1-M4), and a cytoplasmic loop between M3 and M4. The study of structure and dynamics of nAChRs has been motivated by their roles in normal and pathologic neurophysiology, including roles in addiction, neurodegeneration, epilepsy, myasthenia gravis, and congenital myasthenic syndromes (5).Knowledge of the structure of nAChRs has come from biochemical, electrophysiological, and imaging methods (6 -8). These methods, however, do not provide comprehensive structure at atomic resolution. A significant advance toward such information has come recently from the crystallographic structure of acetylcholine-binding protein (AChBP), a water-soluble pentamer that has been the basis for homology modeling of the extracellular...

BackgroundAlong with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites.ResultsComputer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates.ConclusionsInvestigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches.