Associations of Nicotinic Acetylcholine Receptor Subunits Expressed in the Rat Habenula
Kellar, Kenneth J
The goals of this study were to define the nicotinic acetylcholine receptor (nAChR) subtypes expressed in the rat habenula. This was done using three main techniques: single and sequential immunoprecipitation using subunit-specific antibodies, single-point binding with α-conotoxin mII, and saturation binding with [3H]-epibatidine and competition binding with nicotine, cytisine, AT-1001, and sazetidine-A.Single immunoprecipitation experiments revealed that every subunit probed was expressed in the habenula in significant quantities. The α5 subunit was expressed in approximately 26% of nAChRs, the α4 subunit was expressed in approximately 40% of receptors, α3 was expressed in approximately 32% of receptors, β2 was expressed in approximately 48% of receptors, and β4 was expressed in approximately 51% of nAChRs in the habenula. Sequential immunoprecipitations demonstrated that every subunit expressed was significantly associated with every other subunit with one notable exception: the α4 and α5 subunits were not discernably associated in the habenula. Additionally, we found that approximately 20% of nAChRs in the habenula contain both the β2 and β4 subunits. Finally, through binding assays with [3H]-epibatidine and α-conotoxin MII, we determined that there are no detectable α6-containing nAChRs in the habenula.Saturation binding assays determined that nAChRs in the habenula have a Kd for [3H]-epibatidine of 78 pM. Additionally, we found that approximately 20% of nAChRs have a high affinity for Sazetidine-A (a ligand with a high specificity for β2-containing nAChRs) and approximately 70% of nAChRs in the rat habenula had a high affinity for AT-1001 (a ligand with a high specificity for β4-containing nAChRs). From this data, and the data from the immunoprecipitation reactions, we hypothesized that the β2β4* nAChRs in the habenula indicate binding properties much like those of β4-containing nAChRs.Finally, nAChR subunits in the olfactory bulb and olfactory tubercle were probed by single and sequential immunoprecipitation. In the olfactory bulb, the α5 subunit was expressed in approximately 28% of nAChRs, the α4 subunit was expressed in approximately 48% of receptors, α3 was expressed in approximately 41% of receptors, β2 was expressed in approximately 71% of receptors, and β4 was expressed in approximately 33% of nAChRs.
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