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

Controversy surrounds the expression of alpha 7 nicotinic acetylcholine receptors (nAChRs) in adrenal chromaffin cells. In these studies, a7 nAChRs expressed in bovine adrenal chromaffin cells are investigated. Using radiolabeled ligand binding techniques, [ I-125]alpha-bungarotoxin (alpha BGT) 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 alpha BGT was 1.9 nM. These data are consistent with the expression of homomeric 7 alpha nAChRs. Methyllycaconatine (MLA), which binds alpha 7 nAChRs with high affinity, inhibits [ I-121]alpha BGT binding in a concentration-dependent manner with a K-i of 30.6 nM; this value is similar to 10 fold higher than the reported affinity of MLA for alpha 7 nAChRs. We also document the ability of bromoacetylcholine (brACh) to alkylate 07 nAChRs, as has been previous demonstrated for bovine adrenal alpha 3 beta 4 nAChRs. When adrenal nAChRs are immunoprecipated with mAb319, an antibody which recognizes 0 nAChR protein, and then probed with mAb319 using Westem blot analysis, a single band of - 53 kDa is identified. When adrenal nAChRs are immunoprecipated with mAb35, an antibody which recognizes alpha 3 and alpha 5 nAChR proteins, and then probed with mAb3 19 using Western blot analysis, a single band of similar to 53 kDa is identified. Together, these results support the expression of alpha 7 nAChRs in bovine adrenal chrornaffin cells. However, these data suggest that the subunit composition of some of these receptors may include heteromeric alpha 7 nAChRs. (c) 2007 Elsevier Inc. All rights reserved.