Differential alpha 4(+)/(-)beta 2 Agonist-binding Site Contributions to alpha 4 beta 2 Nicotinic Acetylcholine Receptor Function within and between Isoforms

作者：Lucero Linda M; Weltzin Maegan M; Eaton J Brek; Cooper John F; Lindstrom Jon M; Lukas Ronald J; Whiteaker Paul^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2016, 291(5): 2444-2459.

DOI：10.1074/jbc.M115.684373

Two alpha 4 beta 2 nicotinic acetylcholine receptor (alpha 4 beta 2-nAChR) isoforms exist with (alpha 4)(2)(beta 2)(3) and (alpha 4)(3)(beta 2)(2) subunit stoichiometries and high versus low agonist sensitivities (HS and LS), respectively. Both isoforms contain a pair of alpha 4(+)/(-)beta 2 agonist-binding sites. The LS isoform also contains a unique alpha 4(+)/(-)alpha 4 site with lower agonist affinity than the alpha 4(+)/(-)beta 2 sites. However, the relative roles of the conserved alpha 4(+)/(-)beta 2 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 alpha 4 beta 2*-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 beta 2 subunit (-)-face E-loop residues to their non-conserved alpha 4 subunit counterparts or vice versa (beta 2HQT and alpha 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 I-125-mAb 295 binding and was used to define function/nAChR. If the alpha 4(+)/(-)beta 2 sites contribute equally to function, making identical beta 2HQT 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, alpha 4VFL mutation effects were very similar in all positions of both isoforms. Our results indicate that the identity of subunits neighboring the otherwise equivalent alpha 4(+)/(-)beta 2 agonist sites modifies their contributions to nAChR activation and that E-loop residues are an important contributor to this neighbor effect.

• 出版日期2016-1-29