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 NMRspectroscopy. The complete N-terminal extracellular domain is a promising foundation for such models, based on previous studies of alpha 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 alpha 4 and 2 beta subunits with or without the first transmembrane domain ( M1) were expressed in Xenopus oocytes and compared with alpha 4 beta 2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain alpha 4 beta 2 nAChRs formed from subunits with M1 were nearly identical to affinities of alpha 4 beta 2 nAChRs when measured with [ H-3] 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 alpha 4 beta 2 nAChRs. In contrast, [ 3H] epibatidine binding was not detected from the extracellular domain alpha 4 and beta 2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both alpha 4 and beta 2 play an important role for efficient expression of extracellular domain alpha 4 beta 2 nAChRs that are high fidelity structural models of full-length alpha 4 beta 2 nAChRs.