Acta Pharmacologica Sinica (2009) 30: 795-804; doi: 10.1038/aps.2009.68

Aim: α2 nAChR subunit mRNA expression in mice is most intense in the olfactory bulbs and interpeduncular nucleus.  We aimed to investigate the properties of α2* nAChRs in these mouse brain regions.  

Methods: α2 nAChR subunit-null mutant mice were engineered.  Pharmacological and immunoprecipitation studies were used to determine the composition of α2 subunit-containing (α2*) nAChRs in these two regions.  

Results: [¹²⁵I]Epibatidine (200 pmol/L) autoradiography and saturation binding demonstrated that α2 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 β2-selective drug A85380 to inhibit [¹²⁵I]epibatidine binding proved inconclusive, so immunoprecipitation methods were used to further characterize α2* nAChRs.  Protocols were established to immunoprecipitate β2 and β4 nAChRs.  Immunoprecipitation specificity was ascertained using tissue from β2- and β4-null mutant mice, and efficacy was good (>90% of β2* and >80% of β4* nAChRs were routinely recovered).

Conclusion: Immunoprecipitation experiments indicated that interpeduncular nucleus α2* nAChRs predominantly contain β2 subunits, while those in olfactory bulbs contain mainly β4 subunits.  In addition, the immunoprecipitation evidence indicated that both nuclei, but especially the interpeduncular nucleus, express nAChR complexes containing both β2 and β4 subunits.  

Keywords: nicotinic acetylcholine receptors; receptor subtypes; radioligand assay; immunoprecipitation