Neuronal Nicotinic Acetylcholine Receptors on Bovine Chromaffin Cells: Cloning, Expression, and Genomic Organization of Receptor Subunits

Abstract

Neuronal nicotinic acetylcholine receptors from bovine adrenomedullary chromaffin cells play a primary role in triggering catecholamine secretion. In the present study, their constituent subunits were characterized. In addition to the α3 subunit, which we have previously cloned, the presence of α5 and β4 but not of β2 subunits was detected by reverse transcription‐PCR analysis of mRNA from adrenal medulla. In situ hybridization indicated that α3, α5, and β4 subunits are coexpressed in all chromaffin cells. The primary structure of α5 and β4 subunits was determined and functional receptors were obtained upon coinjection of subunit cRNAs into Xenopus oocytes. In contrast to other β4‐containing nicotinic receptors, the ones formed by the bovine β4 subunit are insensitive to the agonist cytisine. Finally, we characterized the intergenic region of α3 and α5 subunits, which together with the β4 subunit, form a gene cluster in rats and chickens. RNase assays and the existence of overlapping cDNAs indicate that, in the bovine genome, the α3 and α5 genes overlap at their 3′ ends. This fact is probably due to inefficient transcription termination, as a result of weak polyadenylation signals.