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 the muscarinic antagonist N-methyl scopolamine (NMS). The apparent molecular mass of the expressed protein, calculated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), is in good agreement with the apparent molecular mass of mAChRs purified from Drosophila brain. Functional expression of the cloned mAChR in this stable cell line has been demonstrated by quantitative fluorescence ratio-imaging of Fura-2-loaded cells. We have observed transient, agonist-induced elevations in intracellular Ca2+ levels which can be completely blocked by atropine, whereas AFDX-116, a muscarinic antagonist which binds preferentially to the vertebrate mAChR M2 subtype, has little effect at 100 mumol l-1. The suitability of this stable Drosophila expression system for the characterization of neurotransmitter receptors is discussed.
Millar
N S
NS
Wellcome Laboratory of Molecular Pharmacology, Department of Pharmacology, University College London, UK.
Baylis
H A
HA
Reaper
C
C
Bunting
R
R
Mason
W T
WT
Sattelle
D B
DB
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D004331:Drosophila melanogaster / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D016257:Fura-2; D005786:Gene Expression Regulation / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D019832:N-Methylscopolamine; D010276:Parasympatholytics / Q000378:metabolism; D010890:Pirenzepine / Q000031:analogs & derivatives / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000096:biosynthesis* / Q000235:genetics; D012602:Scopolamine Derivatives / Q000378:metabolism
D005456:Fluorescent Dyes; D010276:Parasympatholytics; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D010890:Pirenzepine; C048593:otenzepad; D002118:Calcium; D016257:Fura-2; D019832:N-Methylscopolamine