Functional heterologous expression of naturally expressed mouse Î±6âˆ—-nicotinic acetylcholine receptors (mÎ±6âˆ—-nAChRs; where \"âˆ—\" indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mÎ±6mÎ²4hÎ²3- (âˆ¼5-8-fold) or WT mÎ±6mÎ²4mÎ²3-nAChRs (âˆ¼2-fold) yielded higher function than mÎ±6mÎ²4-nAChRs. Function was not detected when mÎ±6 and mÎ²2 subunits were expressed together or in the additional presence of hÎ²3 or mÎ²3 subunits. However, function emerged upon expression of mÎ±6mÎ²2mÎ²3V9â€²S-nAChRs containing Î²3 subunits having gain-of-function V9â€²S (valine to serine at the 9â€²-position) mutations in transmembrane domain II and was further elevated 9-fold when hÎ²3V9â€²Ssubunits were substituted for mÎ²3V9â€²Ssubunits. Studies involving WT or gain-of-function chimeric mouse/human Î²3 subunits narrowed the search for domains that influence functional expression of mÎ±6âˆ—-nAChRs. Using hÎ²3 subunits as templates for site-directed mutagenesis studies, substitution with mÎ²3 subunit residues in extracellular N-terminal domain loops \"C\" (Glu221and Phe223), \"E\" (Ser144and Ser148), and \"Î²2-Î²3\" (Gln94and Glu101) increased function of mÎ±6mÎ²2âˆ—- (â€²2-3-fold) or mÎ±6mÎ²4âˆ—(â€²2-4-fold)-nAChRs. EC50values for nicotine acting at mÎ±6mÎ²4âˆ—-nAChR were unaffected by Î²3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops Î²2-Î²3 and E) interfaces of Î²3 subunits are some of the molecular impediments for functional expression of mÎ±6mÎ²2Î²3- or mÎ±6mÎ²4Î²3-nAChRs.
Journal of Biological Chemistry
Digital Object Identifier (DOI)
Dash, Bhagirathi; Li, Ming D.; and Lukas, Ronald J., "Roles For N-Terminal Extracellular Domains Of Nicotinic Acetylcholine Receptor (Nachr) Î²3 Subunits In Enhanced Functional Expression Of Mouse Î±6Î²2Î²3- And Î±6Î²4Î²3-Nachrs" (2014). Translational Neuroscience. 273.