Lomazzo_2010_J.Neurochem_115_625

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 alpha4 and beta2 subunits. In fact, we identified two populations containing these two predominant subunits, the alpha4beta2 and alpha4beta2alpha5 subtypes which account for approximately 40% and approximately 35%, respectively, of the total [(3)H]EB-labeled receptors. An additional heteromeric subtype with the subunit composition of alpha4beta2alpha3 represented approximately 10% of the total nAChRs, and another 10% of the immunoprecipitated receptors contained alpha4 and beta4 subunits, with or without the alpha3 subunit. To determine if alpha4beta2 and alpha4beta2alpha5 nAChR subtypes differ in their ligand binding affinities, the alpha3- and beta4-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 alpha4beta2 and alpha4beta2alpha5 subtypes. Results suggested these subtypes have comparable binding affinities for the nicotinic ligands used here.