Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1997 International Society for Neurochemistry Neuronal Nicotinic Acetyicholine Receptors on Bovine Chromaffin Cells: Cloning, Expression, and Genomic Organization of Receptor Subunits *A. Campos-Caro, *F. I. Smillie, *E. DomInguez del Toro, 1J. C. Rovira, *F. Vicente-Agulló, *~J. Chapuli, ~J. M. JuIz, §S. Sala, tF. Sala, ~J. J. Ballesta, and *M. Criado Departments of * Neurochemistry, ~Histology, § Physiology, and tPharmacology, and Instituto de Neurociencias, University of Alicante, Alicante, Spain 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 cx3 subunit, which we have previously cloned, the presence of a5 and /94 but not of /32 subunits was detected by reverse transcription—PCR analysis of mRNA from adrenal medulla. In situ hybridization indi- cated that cr3, cr5, and ~34 subunits are coexpressed in all chromaffin cells. The primary structure of a5 and /34 subunits was determined and functional receptors were obtained upon coinjection of subunit cRNAs into Xenopus oocytes. In contrast to other /34-containing nicotinic re- ceptors, the ones formed by the bovine /94 subunit are insensitive to the agonist cytisine. Finally, we character- ized the intergenic region of cr3 and cr5 subunits, which together with the /34 subunit, form a gene cluster in rats and chickens. RNase assays and the existence of over- lapping cDNAs indicate that, in the bovine genome, the cr3 and cr5 genes overlap at their 3’ ends. This fact is probably due to inefficient transcription termination, as a result of weak polyadenylation signals. Key Words: Nico- tinic receptor—Subunits—Expression—Ion channel— Agonist sensitivity—Genes. J. Neurochem. 68, 488—497 (1997). Adrenal chromaffin cells arise developmentally from the neural crest together with other cells such as postganglionic sympathetic neurons (Anderson, 1993). They constitute a relatively homogeneous cell population that perform many of the typical functions of neurons and for this reason are very valuable for approaching fundamental neural phenomena such as neurosecretion (Burgoyne et al., 1993) and neural dif- ferentiation (Unsicker, 1993). Chromaffln cells of the bovine adrenal medulla are cholinergically innervated by the splanchnic nerve from the sympathetic nervous system. Acetylcholine (ACh), released upon stimula- tion of this nerve, activates nicotinic acetylcholine re- ceptors (nAChRs) in chromaffin cells and the subse- quent depolarization triggers catecholamine secretion. nAChRs of neuronal and muscular origin are both pen- tameric oligomers (Cooper et al., 1991; Anand et al., 1991) composed of related subunits that, assembled in various combinations, determine different channel properties (reviewed by Papke, 1993; Karlin and Aka- bas, 1995). Neuronal nAChR subunits are commonly classified as agonist-binding (designated cr2— cr9) and structural (/32— /34) subunits. Functional receptors can be formed by heterologous expression of these subunits either alone (cr7— cr9) or in pairwise combinations (cr2, cr3, or cr4 with either /32 or /34) (reviewed by Sargent, 1993; McGehee and Role, 1995). On the other hand, immunoprecipitation studies have shown that some na- tive nAChRs can be composed of at least three kinds of subunits (Conroy et al., 1992; Vernallis et al., 1993), a fact recently corroborated by expression studies in Xenopus oocytes (RamIrez-Latorre et al., 1996; Wang et al., 1996). Previously, we have cloned the bo- vine a3 (Criado et al., 1992) and cr7 subunits (GarcIa- Guzmán et al., 1995) of neuronal nAChRs, which are expressed in chromaffin cells, probably as components of two different nAChR subtypes. Given that nAChRs assembled from bovine cr7 subunits are blocked by the snake toxin cr-bungarotoxin (GarcIa-Guzmán et al., 1995) and that catecholamine secretion is toxin insen- sitive (Afar et al., 1994), it is reasonable to assume Received June 24, 1996; revised manuscript received September 20, 1996; accepted September 27, 1996. Address correspondence and reprint requests to Dr. M. Criado at Department of Neurochemistry, University of Alicante, Ap. 374, E- 03080 Alicante, Spain. The first two authors contributed equally to this study. The present address of Dr. F. I. Smillie is Wythenshawe Hospital Research Centre, Manchester M23 9LT, U.K. Abbreviations used: ACh, acetyicholine; nAChR, nicotinic acetyl- choline receptor; NFR, normal frog Ringer solution; RT, reverse transcription; SSC, standard saline citrate. 488