# BL-Institut für Neurobiologie, PSF 1860, D-39008 Magdeburg, % Département de Physiologie, CMU, CH-1211 Genève 4,
Nicotinic acetylcholine receptors (nAChRs) are an abundant class of excitatory neurotransmitter receptors in the central nervous system of insects. In Drosophila, five genes encoding potential nAChR subunits have been identified so far and were classified according to their amino-acid sequence as ligand binding a-subunits ALS, Da2/SAD, Da3 and structural subunits ARD, SBD [for review see Gundelfinger E.D., TINS 15, 206 (1992)]. Our aim is to determine the subunit stoichiometry and to functionally characterize the nAChRs in Drosophila which may be regarded as a model system for other insect species. To date, immunoprecipitation experiments indicated that ALS and ARD are components of the same receptor complex that binds a-bungarotoxin (a-Btx) with high affinity [Schloss et al., J Neurochem 75, 1556-1562 (1991)]. Furthermore, immunohistochemical studies with subunit-specific antibodies confirmed the co-distribution of ALS- and ARD-like immunoreactivities (with very few exceptions in the visual system). These ALS/ARD containing receptors are localised in most neuropil regions of the CNS at all developmental stages starting from 10hr-old embryos [Schuster et al., J comp Neurol 335, 149-162 (1993)]. Da2, the second a-like subunit, is similarly distributed, suggesting either a nAChR complex containing two different a-subunits or co-localisation of two receptor subtypes [Jonas et al., J Neurobiol 25, 1494-1508 (1994)]. So far, functional nAChRs were only reconstituted from ALS or Da2 when co-expressed with the chick b2 structural subunit. Using the two microelectrode voltage clamp technique in Xenopus oocytes, the pharmacological profile of these hybrid receptors was determined [Bertrand et al., Europ J Neurosci 6, 859-875 (1994)]. To test whether the two a-subunits can co-assemble into a functional receptor complex with response properties differing from those of ALS/b2 and Da2/b2, mixtures of ALS, Da2 and b2 cDNAs were injected intranuclearly into Xenopus oocytes. Theoretically, at least three different receptor populations could occure after these injections: a) a mixture of two separate receptor populations (ALS/b2 and Da2/b2), b) a mixture of three different receptor types, namely the ALS/b2, Da2/b2 and ALS/Da2/b2 or c) a single type of receptor complex containing the two different a-subunits. Experiments were designed in order to discriminate between these possibilites applying various agonists and antagonists. We found that responses obtained from nAChRs after expression of the mixture significantly differ from ALS/b2 or Da2/b2 alone. These differences cannot be explained by assuming different relative ratios of subtype populations. This strongly supports the idea that the two Drosophila a-subunits co-assemble into the same receptor complex not only in the Xenopus expression system but possibly also in vivo.
Supported by the Kultusministerium of Saxony-Anhalt to E.G. and from the Swiss National Foundation and OFES to D.B.