Pergamon PII S:0028-3908(96) 00087-1 Neuropharmacology, Vol. 35, No. 9/10, pp. 1393–1401, 1996 CopyrightC) 1996 ElsevierScienceLtd. All rightsreserved Printedin Great Britain 0328-3908/96$15.00 +000 Wild-type and Insecticide-resistant Homo-oligomeric GABA Receptors of Drosophila melanogaster Stably Expressed in a Drosophila Cell Line S. D. BUCKINGHAM,’ K. MATSUDA,l A. M. HOSIE,l H. A. BAYLIS,l M. D. SQUIRE,l S. J. LANSDELL,2N. S. MILLAR2AND D. B. SATTELLE1* ‘The BabrahamInstitute Laboratory of Molecular Signaling, Department of Zoology, Universi~ of Cambridge,Downing Street, CambridgeCB23EJ, U.K. and 2WellcomeLaboratory of Molecular Pharmacology,Department of Pharmacology, University College London, Gower Street, London WCIE 6BT, U.K. (Accepted 2 July 1996) Summary-RDL is an monotropic GABA receptor subunit, a product of the Rdl gene, originally identified in the Marylandstrain of Drosophilamelanogaster.Here, we report the generationof a Drosophilamelanogaster cell line (S2-RDLA302s ) stablyexpressinga mutated,dieldrin-resistant(A302S)form of RDL.The propertiesof this dieldrin-resistant,homo-oligomericreceptorhave been comparedwith those of the stably expressed,wild- type form (S2-RDL). Using these stable lines, a striking reduction in sensitivity to both picrotoxinin and dieldrin was observed for responses to GABA of S2-RDLA302s compared to S2-RDL. To determine if these stable insect cell lines generate results similar to those obtained by transient expression in Xenopus laevis oocytes, we have examined the actions of two widely used convulsants, EBOBand TBPS,and a recently developedconvulsantBIDN,on RDL-mediatedGABAresponsesin the two expressionsystems.In both oocytesandS2 cells,thethreeconvulsantsuppressed theamplitudeofresponses to GABA.Thus,in accord withearlierworkonagonistandallostericsites,theS2-RDLcelllineisfoundtoyieldsimilarpharmacological resultsto those obtainedin transientexpressionstudies.Stablecell lines are now availableexpressing susceptibleandresistantformsof anmonotropic receptortargettedby GABAergic insecticides. CopyrightO 1996ElsevierScienceLtd. Keywords—Stable (S2-RDL)celllines,GABAreceptor, Xenopus oocytes, Drosophilamelanogaster, dieldrin resistance. Monotropic receptors for y-aminobutyricacid (GABA) in insects, like those of vertebrates, incorporate an integral chloride ion channel, but show several pharmacological differences from the two classes of vertebrate monotropic GABA receptors (GABAAand GAB&). Unlike verte- brate GABAAreceptors, most insect monotropicGABA receptors are not blocked by bicuculline, but unlike vertebrate GAB* receptors, they are potently activated by isoguvacine(Sattelle et al., 1988;Taylor et al., 1993). Further differencesemerge from studies of the actionsof allosteric modulators. GABA-gated chloride currents in various identified and unidentified insect neurones are enhanced by both 4’-chlorodiazepamand flunitrazepam (Lummis and Sattelle, 1986; Bai, 1994; Aydar et al., 1995), whereas 4’-chlorodiazepamis an antagonist of *To whom correspondence should be addressed (0)1223 336678; fax: +44 (0)1223461954]. [Tel.: +44 vertebrate GABAA receptors. Similarly, barbiturates (Lees et al., 1987) and neuroactive steroids (Rauh et al., 1993) are less active at insect monotropicGABA receptors than at vertebrate GABAA receptors. At the convulsant site, different relative potencies have been revealed in bindingstudies(Sattelle et al., 1991;Cole and Casida, 1992). A detailed understanding of the structural basis of insect monotropicGABA receptors, with their novel pharmacologicalproperties,requires studies on receptors of known molecular composition. Expression studies in cell lines and Xerzopus laevis oocytes of recombinant vertebratemonotropicGABA receptorshave added to our understandingof GABA-gatedchloridechannelfunction, the role of particular subunits and the molecular basis of drug interactions(MacDonaldand Olsen, 1994;Sieghart, 1995). Subunits modified by site-directed mutagenesis and those containing naturally occurring mutations have enabled functionsto be postulatedfor particular subunits 1393