A unique amino acid of the Drosophila GABA receptor with influence on drug sensitivity by two mechanisms.

1. The Drosophila gene Rdl (resistance to dieldrin) encodes a GABA receptor. An alanine-to-serine mutation in this gene at residue 302 confers resistance to cyclodiene insecticides and picrotoxin. Patch clamp analysis of GABA receptors in cultured neurons from wild type and mutant Drosophila was undertaken to investigate the biophysical basis of resistance. 2. In cultured neurons from both wild type and mutant strains, GABA activated a channel that reversed near 0 mV in symmetrical chloride. GABA dose-response characteristics of wild type and mutant receptors were very similar. 3. GABA responses in neurons from the mutant strains showed reduced sensitivity to the GABA antagonists picrotoxin, lindane and t-butyl-bicyclophosphorothionate. Resistance ratios were 116, 970 and 9 for the three blockers, respectively. Inhibition increased with blocker concentration in a manner consistent with saturation of a single binding site. 4. The mutation reduced the single channel conductance by 5% for inward current and 17% for outward current. The single channel current was approximately 60% lower for outward current than for inward current in both wild type and mutant. 5. Open and closed times were both well fitted by the sum of two exponentials. Resistance was associated with longer open times and shorter closed times, reflecting a net stabilization of the channel open state by a factor of approximately five. 6. The mutation was associated with a marked reduction in the rate of GABA-induced desensitization, and a net destabilization of the desensitized conformation by a factor of 29. 7. The Rdl mutation manifests resistance through two different mechanisms. (a) The mutation weakens drug binding to the antagonist-favoured (desensitized) conformation by a structural change at the drug binding site. (b) The mutation destabilizes the antagonist-favoured conformation in an allosteric sense. The global association of a single amino acid replacement with cyclodiene resistance suggests that the resistance phenotype depends on changes in both of these properties, and that insecticides have selected residue 302 of Rdl for replacement because of its unique ability to influence both of these functions. 8. The location of alanine 302 in the sequence of the Rdl gene product supports a mechanism of action in which convulsants such as picrotoxin bind within the channel lumen, where they induce a rapid conformational change to the desensitized state.