Vertebrate GABA-A receptors are complex hetero-multimers formed from a number of different subunits whose distribution in the nervous system has been well described. GABA receptors have been implicated in a number of human disorders and bursting responses from picrotoxinin (PTX) have been used as a model of epilepsy. However, many central questions relating to GABA receptor structure/function remain unanswered. The investigator recently identified a novel GABA receptor subunit gene Rdl (Resistance to dieldrin) that was cloned from a Drosophila mutant resistant to PTX and cyclodiene insecticides. He has functionally expressed in Xenopus oocytes GABA-gated chloride channels composed of homomultimers of Rdl and rescued the susceptible phenotype by P element mediated germline transformation. He has identified a single amino acid substitution (alanine 302>serine) in Rdl, which confers resistance to cyclodienes and PTX. The applicant now proposes to investigate the structure/function of the receptors that contain this novel subunit and to use this gene as a model system to answer questions central to GABA receptor biology. This proposal addresses a number of specific questions. (1) Is the presence of alanine302 critical for drug binding and what are the effects of different amino acid replacements at this residue? Prior work suggests that the A302>S replacement confers drug resistance via a novel dual mechanism involving both direct changes in the drug binding site and allosteric destabilization of the drug- preferred desensitized state of the receptor. To test this hypothesis, the investigator proposes to examine the effect of the second resistance associated replacement Ala302>Gly and a series of other side chain replacements within this model framework to look for differential effects on the rate of receptor desensitization and drug binding. It is also proposed to examine the effects of replacements that are expected only to cause allosteric effects on drug resistance. Secondly: Is Rdl expressed as a homo- or hetero-multimer? The investigator proposes three approaches to examine this question. (1) Coexpression of Rdl with the Drosophila beta subunit GABA-A receptor homolog to look for evidence of hetermultimeric assembly. Support for coassembly of these two subunits comes from preliminary studies in which the investigator has identified a novel additional PTX insensitive, but bicucullin sensitive response to prolonged application of GABA, which corresponds to channels containing both Rdl and beta subunits. (2) Use of Rdl and beta subunit specific antibodies in both immunohistochemistry and immunopurification studies. His preliminary immunohistochemical results in Drosophila embryos demonstrate specific patterns of staining for the two different receptor subunits in the CNS. In the ventral ganglion Rdl appears to be confined to the longitudinal commissures whereas the beta subunit stains in the cell bodies surrounding the ganglia. The investigator proposes to conduct detailed sequential and double-labeling experiments to test whether the two subunits are coexpressed during different lifestages and/or in different tissues. He has also performed preliminary immunopurification studies using the Rdl-specific antibody to isolate native GABA receptors from fly head homogenates. The results of these studies suggest that receptors purified using this subunit antibody also contain beta subunits, supporting the hypothesis that Rdl and beta subunits coassemble in the Drosophila nervous system. (3) Examination of the conductance of in vivo receptors formed from Rdl transgenes of lowered conductance in an Rdl null background. Following the introduction of a P-element introduced transgene with lowered conductance into Rdl null flies (homozygous for inversions disrupting Rdl), the investigator proposes to examine the conductance of Rdl containing receptors in vivo. If the receptors from these mutants show the same lowered conductance as the transgene, this will support the null hypothesis that Rdl is a homomultimer. Finally: Does Rdl interact with any other GABA receptor subunit genes? Three different genetic screens will be used to search for genes interacting with Rdl: (A) screens for suppressors or enhancers of the resistance phenotype, (B) screens for a putative lindane receptor mutant; and (C) P-element site directed mutagenesis of known chloride ion channel genes.
| Brotz, T M; Bochenek, B; Aronstein, K et al. (1997) gamma-Aminobutyric acid receptor distribution in the mushroom bodies of a fly (Calliphora erythrocephala): a functional subdivision of Kenyon cells? J Comp Neurol 383:42-8 |
