Pharmacological and immunochemical characterization of alpha 2*nicotinic acetylcholine receptors (nAChRs) in mouse brain

Aim: alpha 2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of alpha 2* nAChRs in these mouse brain regions. Methods: alpha 2 nAChR subunit-null mutant mice were engineered. Pharmacological and immunoprecipitation studies were used to determine the composition of alpha 2 subunit-containing (alpha 2*) nAChRs in these two regions. Results: [I-125] Epibatidine (200 pmol/L) autoradiography and saturation binding demonstrated that alpha 2 deletion reduces nAChR expression in both olfactory bulbs and interpeduncular nucleus (by 4.8 +/- 1.7 and 92 +/- 26 fmol.mg(-1) protein, respectively). Pharmacological characterization using the beta(2)-selective drug A85380 to inhibit [I-125] epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize alpha 2* nAChRs. Protocols were established to immunoprecipitate beta 2 and beta 4 nAChRs. Immunoprecipitation specificity was ascertained using tissue from beta 2- and beta 4-null mutant mice, and efficacy was good (>90% of beta 2* and >80% of beta 4* nAChRs were routinely recovered). Conclusion: Immunoprecipitation experiments indicated that interpeduncular nucleus alpha 2* nAChRs predominantly contain beta 2 subunits, while those in olfactory bulbs contain mainly beta 4 subunits. In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both beta 2 and beta 4 subunits.