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J. Biol. Chem., Vol. 280, Issue 48, 39990-40002, December 2, 2005

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Extracellular Domain Nicotinic Acetylcholine Receptors Formed by 4 and 2 Subunits^*

Alexandra M. Person, Kathy L. Bills, Hong Liu, Shaleen K. Botting, Jon Lindstrom, and Gregg B. Wells1

From the Department of Pathology and Laboratory Medicine, College of Medicine, Texas A&M University System Health Science Center, College Station, Texas 77843-1114 and the Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104-6074

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 42 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain 42 nAChRs formed from subunits with M1 were nearly identical to affinities of 42 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 42 nAChRs. In contrast, [³H]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 42 nAChRs that are high fidelity structural models of full-length 42 nAChRs.

Received for publication, May 9, 2005 , and in revised form, August 4, 2005.

^* This work was supported by National Institutes of Health Grants NS01903 and NS11323, Philip Morris U. S. A. Inc., Philip Morris International, and the Smokeless Tobacco Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, Texas A&M University System Health Science Center, 208 Reynolds Medical Bldg., 1114 TAMU, College Station, TX 77843-1114. Tel.: 979-458-8888; Fax: 979-862-1299; E-mail: gbwells{at}tamu.edu.

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