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The Journal of Neuroscience, October 8, 2003, 23(27):9004-9015
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Cellular/Molecular
Human  4 2 Acetylcholine Receptors Formed from Linked Subunits
Yan Zhou,
Mark E. Nelson,
Alexander Kuryatov,
Catherine Choi,
John Cooper, and
Jon Lindstrom
Department of Neuroscience, University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104-6074
We prepared concatamers of  4 and  2 subunits for human nicotinic acetylcholine receptors (AChRs), in which the C terminus of 4 was linked to the N terminus of 2, or vice versa, via a tripeptide sequence repeated 6 or 12 times, and expressed them in Xenopus oocytes. Linkage did not substantially alter channel amplitude or channel open-duration. Linkage at the C terminus of 4 prevented AChR potentiation by 17--estradiol by disruption of its binding site. Assembly of AChRs from concatamers was less efficient, but function was much more efficient than that of unlinked subunits. With both linked and free subunits, greater ACh-induced currents per surface AChR were observed with the (4)3(2)2 stoichiometry than with the (4)2(2)3 stoichiometry. The (4)3(2)2 stoichiometry exhibited much lower ACh sensitivity. When concatamers were expressed alone, dipentameric AChRs were formed in which the (4)2(2)3 pentamer was linked to the (4)3(2)2 pentamer. Dipentamers were selectively expressed on the cell surface, whereas most monopentamers with dangling subunits were retained intracellularly. Coexpression of concatamers with monomeric 2, 4, or 4 subunits resulted in monopentamers, the stoichiometry of which was determined by the free subunit added. Linkage between the C terminus of 2 and the N terminus of 4 favored formation of ACh-binding sites within the concatamer, whereas linkage between the C terminus of 4 and the N terminus of 2 favored formation of ACh-binding sites between concatamers. These protein-engineering studies provide insight into the structure and function of 42 AChRs, emphasizing the functional differences between 42 AChRs of different stoichiometries.
Key words: concatamer; nicotinic; receptor; Xenopus oocyte; stoichiometry; efficacy
Received July 7, 2003;
revised August 11, 2003;
accepted August 11, 2003.
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