Extracellular Domain Nicotinic Acetylcholine Receptors Formed by alpha 4 and beta 2 Subunits

• Published in: The Journal of biological chemistry Vol. 280; no. 48; pp. 39990 - 40002
• Main Authors: Person, Alexandra M, Bills, Kathy L, Liu, Hong, Botting, Shaleen K, Lindstrom, Jon, Wells, Gregg B
• Format: Journal Article
• Language: English
• Published: 02.12.2005
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M505087200

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 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 beta 2 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 [ super(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 alpha 4 beta 2 nAChRs. In contrast, [ super(3)H]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.