Disease relevance of Rdl

• Recombinant baculoviruses containing two alternative splice forms of the Drosophila Rdl GABA receptor gene were constructed [1].
• In order to examine the conservation of the mechanism of cyclodiene insecticide resistance between species we cloned a cDNA from the yellow fever mosquito Aedes aegypti homologous to the resistance gene Rdl in Drosophila [2].
• Thus, the selective toxicity of fipronil and fipronil-derived residues is due in part to the higher potency of the parent compound at the insect versus the mammalian GABA receptor but is also dependent on the relative rates of conversion to the more persistent and less selective sulfone metabolite and desulfinyl photoproduct [3].
• Properties of GABA-induced current in cultured CNS (ganglion) neurons of cutworm moths (Spodoptera litura) were studied using a whole-cell patch-clamp technique [4].

Psychiatry related information on Rdl

• Recovery of normal locomotor activity and the righting reflex in DABA- and NipA-treated flies was achieved by coadministration of bicuculline (BIC), a GABA receptor antagonist that supresses GABAergic activity in mammals [5].
• A missense mutation has been found in the gene encoding the GABA-A beta 3 subunit in a patient with chronic insomnia [6].
• However, the properties of GABAergic neurons and the cellular effects of GABA have not been described in Drosophila, an important model organism for olfaction research [7].

High impact information on Rdl

• Subsequently, genetic analysis and metabolite profiling confirmed a complex role for ornithine and GABA levels in modification of survival time upon chronic nicotine exposure [8].
• GABAA receptors in vertebrates assemble as heteromultimers to form an integral chloride ion channel [9].
• A point mutation in a Drosophila GABA receptor confers insecticide resistance [9].
• Picrotoxinin (PTX) and cyclodiene insecticides are GABAA receptor antagonists which competitively displace each other from the same binding site [9].
• This mutation not only identifies a single amino acid conferring high levels of resistance to the important GABA receptor antagonist PTX but also, by conferring resistance to cyclodienes, may account for over 60% of reported cases of insecticide resistance [9].

Biological context of Rdl

• The Drosophila gamma-aminobutyric acid (GABA) receptor subunit gene Rdl was isolated on the basis of a mutant phenotype showing high levels of insensitivity to picrotoxinin and cyclodiene insecticides [10].
• Following analysis of two dissimilar cDNAs isolated from the locus, we report that Rdl undergoes extensive alternative splicing at two locations in the putative extracellular domain [10].
• GABA receptors containing Rdl subunits mediate fast inhibitory synaptic transmission in Drosophila neurons [11].
• Amino acid sequence analysis of a cDNA from the locus reveals homology with vertebrate GABAA subunits [12].
• Resistance is due to insensitivity of the cyclodiene/picrotoxinin binding site on the gamma-aminobutyric acid subtype A (GABAA) receptor-chloride ionophore complex [12].

Anatomical context of Rdl

• When expressed in Xenopus laevis oocytes, the subunit functions efficiently, presumably as a homooligomeric complex and is activated by GABA or muscimol [13].
• To our knowledge, this represents the first cloning of an invertebrate GABA receptor and also allows us to manipulate the resistance status of an insect via germ-line transformation [12].
• A cloned Drosophila gamma-aminobutyric acid GABA receptor subunit (Rdl) has been stably expressed as a functional homo-oligomeric ion channel in a Drosophila cell line [14].
• Rdl is expressed in both the embryonic central and peripheral nervous system [15].
• In contrast, Rdl receptor subunits appear confined to the neuropil in all developmental stages [16].

Associations of Rdl with chemical compounds

• Drosophila gamma-aminobutyric acid receptor gene Rdl shows extensive alternative splicing [10].
• Rdl codes for a subunit of a novel class of GABA gated chloride ion channels and resistance is correlated with replacement of the same amino acid residue in a wide range of species from different insect orders [17].
• Pentobarbitone greatly enhances GABA-evoked currents, whereas the neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one demonstrates a large reduction in both the potency and maximal effect when compared with its actions upon vertebrate GABA type A receptors [13].
• Of the four receptor variants tested, the double mutant showed the highest resistance to fipronil, following repeated GABA applications [18].
• In addition to providing a possible physiological role for the alternative splicing of Rdl, these data delineate a hitherto functionally unassigned region of the N-terminal domain of GABA receptor subunits, which affects agonist potency and aligns closely with known determinants of potency in nicotinic acetylcholine receptors [19].

Other interactions of Rdl

• Interestingly, despite the complexity of the encoded receptors or enzymes, very few amino acid residues are replaced in different resistant insects: one within RDL, two within PARA and three or more within AChE [20].
• In addition, we demonstrated a direct correlation between the amount of dFmrp and the expression of the GABA receptor subunits Rdl and Grd [21].
• Mutation of the human beta3 subunit asparagine to methionine (beta3 N289M found in the equivalent position in Rdl completely inhibited both the GABA-modulatory and GABA-mimetic action of etomidate (10-300 microM) acting at alpha6beta3 N289Mgamma2L receptors [22].
• It has been shown that DBI and its processing products are involved in various specific biological processes such as GABAA/benzodiazepine receptor modulation, acyl coenzyme A metabolism, steroidogenesis, and insulin secretion [23].
• This approach yielded three discrete amplified sequence elements (designated LCCH1, LCCH2, and LCCH3) that contained open reading frames and > 40% amino acid sequence identity to the corresponding regions of vertebrate ligand-gated chloride channel genes [24].

Analytical, diagnostic and therapeutic context of Rdl

• Molecular cloning of the cDNA encoding the RDL GABA receptor subunit of this strain revealed the presence of two mutations: the Rdl mutation (A301G) and an additional mutation in the third transmembrane domain (T350M) [18].
• Genomic clones from the Rdl locus of Drosophila, whose mutant phenotype is resistant to cyclodiene insecticides and picrotoxin, were characterized by restriction mapping and partial sequencing to determine intron/exon structure [25].
• Detailed immunocytochemistry of Drosophila brain sections showed particularly strong anti-Rdl antibody staining in the optic lobes, ellipsoid body, fan shaped body, ventrolateral protocerebrum and the glomeruli of the antennal lobes [26].
• In situ hybridization using Rdl cDNA probes and the anti-Rdl antibody shows that Rdl message and protein are highly expressed in the developing central nervous system (CNS) of 15-17 h embryos [26].
• We confirmed such GABA control mechanisms in an independent model system, i.e. primary Drosophila cell cultures raised in elevated [K+] [27].

References