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 indicated that cr3, cr5, and~34subunits 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 receptors, the ones formed by the bovine /94 subunit are insensitive to the agonist cytisine. Finally, we characterized 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 overlapping 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.

An aspartate residue in the M2-M3 loop of neuronal nicotinic receptor K K U subunits is a major determinant of the channel functional response. This residue is conserved in most L L R subunits, e.g. human and rat, but not in others, e.g. bovine. We have used these differences to examine the mechanism by which this residue alters the functional properties of K K Q L L R receptors. Having ruled out an effect on the macroscopic binding ability of the agonist, the level of receptor expression, or the single channel conductance, the results suggest that receptors lacking that residue have a deficient coupling between binding and gating.z 1998 Federation of European Biochemical Societies.

We have examined the role of a highly conserved arginine (R209), which flanks the M1 transmembrane segment of nAChRs, in the biogenesis and function of neuronal nAChRs. Point mutations revealed that, in alphaBgtx-sensitive neuronal alpha7 nAChRs, the conserved arginine is required for the transport of assembled receptors to the cell surface. By contrast, R209 does not play any role in the transport of assembled alpha-Bgtx-insensitive neuronal alpha3beta4 nAChRs to the cell surface. However, a basic residue at this position of alpha3 and beta4 subunits is necessary for either synthesis, folding, or assembly of alpha3beta4 receptors. Moreover, electrophysiological experiments revealed that in alpha3beta4 receptors the conserved arginine of the alpha3 subunit is involved in either coupling agonist binding to the channel or regulating single channel kinetics.