Pharmacological and immunological identification of native α7 nicotinic receptors: Evidence for homomeric and heteromeric α7 receptors

Abstract

Controversy surrounds the expression of α7 nicotinic acetylcholine receptors (nAChRs) in adrenal chromaffin cells. In these studies, α7 nAChRs expressed in bovine adrenal chromaffin cells are investigated. Using radiolabeled ligand binding techniques, [ 125I]α-bungarotoxin (αBGT) binding reaches equilibrium within 4 h and is saturable with a K d value of 4.2 nM. Using homologous competition experiments, the K i for binding of αBGT was 1.9 nM. These data are consistent with the expression of homomeric α7 nAChRs. Methyllycaconatine (MLA), which binds α7 nAChRs with high affinity, inhibits [ 125I]αBGT binding in a concentration-dependent manner with a K i of 30.6 nM; this value is ∼ 10 fold higher than the reported affinity of MLA for α7 nAChRs. We also document the ability of bromoacetylcholine (brACh) to alkylate α7 nAChRs, as has been previous demonstrated for bovine adrenal α3β4 nAChRs. When adrenal nAChRs are immunoprecipated with mAb319, an antibody which recognizes α7 nAChR protein, and then probed with mAb319 using Western blot analysis, a single band of ∼ 53 kDa is identified. When adrenal nAChRs are immunoprecipated with mAb35, an antibody which recognizes α3 and α5 nAChR proteins, and then probed with mAb319 using Western blot analysis, a single band of ∼ 53 kDa is identified. Together, these results support the expression of α7 nAChRs in bovine adrenal chromaffin cells. However, these data suggest that the subunit composition of some of these receptors may include heteromeric α7 nAChRs.