Quantitative analysis of the heteromeric neuronal nicotinic receptors in the rat hippocampus

The objective of this study was to identify and quantify the heteromeric neuronal nicotinic receptors (nAChRs) in the rat hippocampus. The density of nAChR subtypes was assessed by labeling them with [(3)H]epibatidine ([(3)H]EB) followed by immunoprecipitation with subunit-selective antibodies. Sequential immunoprecipitation assays were used to establish associations between two different subunits, which then allowed the full subunit composition of the receptors to be deduced. Our results show that most of the hippocampal heteromeric nAChRs contain α4 and β2 subunits. In fact, we identified two populations containing these two predominant subunits, the α4β2 and α4β2α5 subtypes which account for ∼ 40% and ∼ 35%, respectively, of the total [(3)H]EB-labeled receptors. An additional heteromeric subtype with the subunit composition of α4β2α3 represented ∼ 10% of the total nAChRs, and another 10% of the immunoprecipitated receptors contained α4 and β4 subunits, with or without the α3 subunit. To determine if α4β2 and α4β2α5 nAChR subtypes differ in their ligand binding affinities, the α3- and β4-containing receptors were first removed by immunoprecipitation and then, competition studies with acetylcholine, nicotine, cytisine and sazetidine-A against [(3)H]EB were carried out on the remaining α4β2 and α4β2α5 subtypes. Results suggested these subtypes have comparable binding affinities for the nicotinic ligands used here.