Expression of a Drosophila GABA receptor in a baculovirus insect cell system. Functional expression of insecticide susceptible and resistant GABA receptors from the cyclodiene resistance gene Rdl.
Recombinant baculoviruses containing two alternative splice forms of the Drosophila Rdl GABA receptor gene were constructed. Spodoptera frugiperda (Sf21) cells infected with either splice form expressed a transcript of expected size (2.5 kb). Western blotting of cell membrane extracts and immunoprecipitation experiments with an anti-Rdl antiserum recognized a protein of the expected size of approximately 65 kDa. Whole cell patch clamp analysis of cells infected with either splice form revealed functional expression of GABA gated chloride ion channels which were blocked by application of 1 microM picrotoxinin. Following replacement of alanine 302 with a serine, a mutation associated with resistance to picrotoxinin and cyclodiene insecticides, mutant channels showed similar levels of insensitivity to picrotoxinin (approximately 100-fold) as those observed in recordings from cultured Drosophila neurons. The significance of the expression of an insect GABA receptor in an insect cell line and the similarity of the results from these functional expression studies to recordings from cultured neurons is discussed.
Lee
H J
HJ
Department of Entomology, University of Wisconsin-Madison 53706.
Rocheleau
T
T
Zhang
H G
HG
Jackson
M B
MB
ffrench-Constant
R H
RH
D017398:Alternative Splicing; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D004330:Drosophila; D029721:Drosophila Proteins; D007305:Insecticide Resistance / Q000235:genetics; D008564:Membrane Potentials; D009036:Moths; D009154:Mutation; D010852:Picrotoxin / Q000494:pharmacology; D011963:Receptors, GABA-A / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*
D029721:Drosophila Proteins; D010852:Picrotoxin; D011963:Receptors, GABA-A; C082030:Rdl protein, Drosophila