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

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

SUBMITTER: Lucero LM 

PROVIDER: S-EPMC4732226 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature