Abstract
AIM: alpha2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus. We aimed to investigate the properties of alpha2* nAChRs in these mouse brain regions. METHODS: alpha2 nAChR subunit-null mutant mice were engineered. Pharmacological and immunoprecipitation studies were used to determine the composition of alpha2 subunit-containing (alpha2*) nAChRs in these two regions. RESULTS: [(125)I]Epibatidine (200 pmol/L) autoradiography and saturation binding demonstrated that alpha2 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 beta2-selective drug A85380 to inhibit [(125)I]epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize alpha2* nAChRs. Protocols were established to immunoprecipitate beta2 and beta4 nAChRs. Immunoprecipitation specificity was ascertained using tissue from beta2- and beta4-null mutant mice, and efficacy was good (>90% of beta2* and >80% of beta4* nAChRs were routinely recovered). CONCLUSION: Immunoprecipitation experiments indicated that interpeduncular nucleus alpha2* nAChRs predominantly contain beta2 subunits, while those in olfactory bulbs contain mainly beta4 subunits. In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both beta2 and beta4 subunits.