The effect of a transmembrane amino acid on etomidate sensitivity of an invertebrate GABA receptor

The γ‐aminobutyric acid (GABA)‐modulatory and GABA‐mimetic actions of etomidate at mammalian GABA A receptors are favoured by β 2 ‐ or β 3 ‐ versus β 1 ‐subunit containing receptors, a selectivity which resides with a single transmembrane amino acid (β 2 N290 , β 3 N289 , β 1 S290 ). Here, we have utilized the Xenopus laevis oocyte expression system in conjunction with the two‐point voltage clamp technique to determine the influence of the equivalent amino acid (M314) on the actions of this anaesthetic at an etomidate‐insensitive invertebrate GABA receptor ( Rdl ) of Drosophila melanogaster . Complementary RNA‐injected oocytes expressing the wild type Rdl GABA receptor and voltage‐clamped at −60 mV responded to bath applied GABA with a concentration‐dependent inward current response and a calculated EC 50 for GABA of 20±0.4 μ M . Receptors in which the transmembrane methionine residue (M314) had been exchanged for an asparagine ( Rdl M314N ) or a serine ( Rdl M314S ) also exhibited a concentration‐dependent inward current response to GABA, but in both cases with a reduced EC 50 of 4.8±0.2 μ M . Utilizing the appropriate GABA EC 10 , etomidate (300 μ M ) had little effect on the agonist‐evoked current of the wild type Rdl receptor. By contrast, at Rdl M314N receptors, etomidate produced a clear concentration‐dependent enhancement of GABA‐evoked currents with a calculated EC 50 of 64±3 μ M and an E max of 68±2% (of the maximum response to GABA). The actions of etomidate at Rdl M314N receptors exhibited an enantioselectivity common to that found for mammalian receptors, with 100 μ M R ‐(+)‐etomidate and S ‐(−)‐etomidate enhancing the current induced by GABA (EC 10 ) to 52±6% and 12±1% of the GABA maximum respectively. The effects of this mutation were selective for etomidate as the GABA‐modulatory actions of 1 m M pentobarbitone at wild type Rdl (49±4% of the GABA maximum) and Rdl M314N receptors (53±2% of the GABA maximum) were similar. Additionally, the modest potentiation of GABA produced by the anaesthetic neurosteroid 5α‐pregnan‐3α‐ol‐20‐one ( Rdl =25±4% of the GABA maximum) was not altered by this mutation ( Rdl M314N =18±3% of the GABA maximum). Etomidate acting at β 1 (S290)‐containing mammalian GABA A receptors is known to produce only a modest GABA‐modulatory effect. Similarly, etomidate acting at Rdl M314S receptors produced an enhancement of GABA but the magnitude of the effect was reduced compared to Rdl M314N receptors. Etomidate acting at human α 6 β 3 γ 2L receptors is known to produce a large enhancement of GABA‐evoked currents and at higher concentrations this anaesthetic directly activates the GABA A receptor complex. Mutation of the human β 3 subunit asparagine to methionine (β 3 N289M found in the equivalent position in Rdl completely inhibited both the GABA‐modulatory and GABA‐mimetic action of etomidate (10–300 μ M ) acting at α 6 β 3 N289M γ 2L receptors. It was concluded that, although invertebrate and mammalian proteins exhibit limited sequence homology, allosteric modification of their function by etomidate can be influenced in a complementary manner by a single amino acid substitution. The results are discussed in relation to whether this amino acid contributes to the anaesthetic binding site, or is essential for transduction. Furthermore, this study provides a clear example of the specificity of anaesthetic action.