Extracellular Domain Nicotinic Acetylcholine Receptors Formed by α4 and β2 Subunits

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 α7 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 α4 and β2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with α4β2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain α4β2 nAChRs formed from subunits with M1 were nearly identical to affinities of α4β2 nAChRs when measured with [3H]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 α4β2 nAChRs. In contrast, [3H]epibatidine binding was not detected from the extracellular domain α4 and β2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both α4 and β2 play an important role for efficient expression of extracellular domain α4β2 nAChRs that are high fidelity structural models of full-length α4β2 nAChRs.