Two Î±4Î²2 nicotinic acetylcholine receptor (Î±4Î²2-nAChR) isoforms exist with (Î±4)2(Î²2)3 and (Î±4)2(Î²2)3 subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of Î±4(+)/(-)Î²2 agonist- binding sites. The LS isoform also contains a unique Î±4(+)/(-)Î±4 site with lower agonist affinity than the Î±4(+)/(-)Î²2 sites. However, the relative roles of the conserved Î±4(+)/(-)Î²2 agonist-binding sites in and between the isoforms have not been studied. We used a fully linked subunit concatemeric nAChR approach to express pure populations of HS or LS isoform Î±4Î²2âˆ—-nAChR. This approach also allowed us to mutate individual subunit interfaces, or combinations thereof, on each isoform background. We used this approach to systematically mutate a triplet of Î²2 subunit (-)-face E-loop residues to their non-conserved Î±4 subunit counterparts or vice versa (Î²2HQT and Î±4VFL, respectively). Mutant-nAChR constructs (and unmodified controls) were expressed in Xenopus oocytes. Acetylcholine concentration-response curves and maximum function were measured using two-electrode voltage clamp electrophysiology. Surface expression was measured with 125I-mAb 295 binding and was used to define function/nAChR. If the Î±4(+)/(-)Î²2 sites contribute equally to function, making identical Î²2HQT substitutions at either site should produce similar functional outcomes. Instead, highly differential outcomes within the HS isoform, and between the two isoforms, were observed. In contrast, Î±4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent Î±4(+)/(-)Î²2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.
Journal of Biological Chemistry
Digital Object Identifier (DOI)
Lucero, Linda M.; Weltzin, Maegan M.; Eaton, J. Brek; Cooper, John F.; Lindstrom, Jon M.; Lukas, Ronald J.; and Whiteaker, Paul, "Differential Î±4(+)/(-)Î²2 Agonist-Binding Site Contributions To Î±4Î²2 Nicotinic Acetylcholine Receptor Function Within And Between Isoforms" (2016). Translational Neuroscience. 202.