Agonist pharmacology of two Drosophila GABA receptor splice variants

Abstract

1 The Drosophila melanogaster y-aminobutyric acid (GABA) receptor subunits, RDLaC and DRC 17-1-2, form functional homo-oligomeric receptors when heterologously expressed in Xenopus laevis oocytes. The subunits differ in only 17 amino acids, principally in regions of the N-terminal domain which determine agonist pharmacology in vertebrate ionotropic neurotransmitter receptors. A range of conformationally restricted GABA analogues were tested on the two homo-oligomers and their agonist pharmacology compared with that of insect and vertebrate iontropic GABA receptors. 2 The actions of GABA, isoguvacine and isonipecotic acid on RDLaC and DRC 17-1-2 homo-oligomers were compared, by use of two-electrode voltage-clamp. All three compounds were full agonists of both receptors, but were 4-6 fold less potent agonists of DRC 17-1-2 homo-oligomers than of RDLaC. However, the relative potencies of these agonists on each receptor were very similar.3 A more complete agonist profile was established for RDLac homo-oligomers. The most potent agonists of these receptors were GABA, muscimol and trans-aminocrotonic acid (TACA), which were approximately equipotent. RDLaC homo-oligomers were fully activated by a range of GABA analogues, with the order of potency: GABA> ZAPA ((Z)-3-[(aminoiminomethyl)thio]prop-2-enoic acid)> isoguvacine > imidazole-4-acetic acid > isonipecotic acid > cis-aminocrotonic acid (CACA) > P-alanine. 3-Aminopropane sulphonic acid (3-APS), a partial agonist of RDLLC homo-oligomers, was the weakest agonist tested and 100 fold less potent than GABA. 4 SR95531, an antagonist of vertebrate GABAA receptors, competitively inhibited the GABA responses of RDLaC homo-oligomers, which have previously been found to be insensitive to bicuculline. However, its potency (IC50 500 gM) was much reduced when compared to GABAA receptors.5 The agonist pharmacology of Drosophila RDLac homo-oligomers exhibits aspects of the characteristic pharmacology of certain native insect GABA receptors which distinguish them from vertebrate GABA receptors. The high potency and efficacy of isoguvacine and ZAPA distinguishes RDLIC homo-oligomers from bicuculline-insensitive vertebrate GABAC receptors, while the low potency of SR95531 and 3-APS distinguishes them from GABAA receptors. The differences in the potency of agonists on RDLaC and DRC 17-1-2 homo-oligomers observed in the present study may assist in identification of further molecular determinants of GABA receptor function.

Insect GABA Receptors: Splicing, Editing, and Targeting by Antiparasitics and Insecticides

“…Such pharmacology is typical of many native insect GABARs Sattelle, 1985, 1986;Sattelle et al, 1988). Unlike many native insect GABARs (Brotz and Borst, 1996), however, RDL is sensitive to CACA, an agonist of vertebrate GABA C , but not of GABA A , receptors (Hosie and Sattelle, 1996a). However, it should be noted that CACA does act on some insect GABARs, including those that mediate inhibition by identified filiform hair receptors of an identified projection interneuron (Gauglitz and Pfluger, 2001).…”

“…The exon 6 alternatively spliced residues of RDL lie in loops F and C (Fig. 1B) and exon 6 splicing affects the expressed receptor's affinity for GABA (Hosie and Sattelle, 1996a) (Fig. 1C).…”

“…RDL homomeric receptors are distinguished from vertebrate GABA A receptors by their insensitivity to bicuculline, their low sensitivity to 3-aminopropanesulfonic acid, and their high sensitivity to both isoguvacine (Hosie and Sattelle, 1996a) and muscimol (Buckingham et al, 1994a) (Fig. 1C).…”

“…1A), all of which are expressed (ffrench-Constant and Rocheleau, 1993). Three splice variants (RDLac, RDLbd, and RDLab) have been cloned and functionally expressed (Hosie and Sattelle, 1996a). Residues that vary in exon 3 lie upstream to loop D of the GABA binding site and near the equivalent positions of known determinants of agonist potency in GABA A receptors of vertebrates.…”

Synthesis of 1,3-di- and 1,3,4-trisubstituted 1,6-dihydro-6-iminopyridazines as competitive antagonists of insect GABA receptors

“…Whereas bicuculline is inactive against most insect GABARs, gabazine shows weak or moderate antagonistic activity against insect GABARs. [26][27][28] It has been previously reported that the introduction of substituents at the 5-position of the pyridazine ring of gabazine produces competitive antagonists with micromolar-affinity against the parasitic nematode Ascaris suum, whereas the 5-substitutent is detrimental to rat GABARs. 29,30) This selectivity is informative in terms of the design of insecticides.…”