FUNCTIONAL EXPRESSION OF A CLONED DROSOPHILA MUSCARINIC ACETYLCHOLINE-RECEPTOR IN A STABLE DROSOPHILA CELL-LINE

A cloned Drosophila muscarinic acetylcholine receptor (mAChR) has been stably expressed in a Drosophila cell line (S2) under the control of an inducible Drosophila metallothionein promoter, A clonal cell line ( S2-Dm1-1) has been isolated which, after induction of mAChR expression with CuSO4, exhibits high-affinity, saturable, specific binding of th e muscarinic antagonist N-methyl scopolamine (NMS). The apparent molec ular mass of the expressed protein, calculated by sodium dodecyl sulph ate-polyacrylamide gel electrophoresis (SDS-PAGE), is in good agreemen t with the apparent molecular mass of mAChRs purified from Drosophila brain. Functional expression of the cloned mAChR in this stable cell l ine has been demonstrated by quantitative fluorescence ratio-imaging o f Fura-2-loaded cells, We have observed transient, agonist-induced ele vations in intracellular Ca2+ levels which can be completely blocked b y atropine, whereas AFDX-116, a muscarinic antagonist which binds pref erentially to the vertebrate mAChR Mt subtype, has little effect at 10 0 mu moll(-1). The suitability of this stable Drosophila expression sy stem for the characterization of neurotransmitter receptors is discuss ed.