THE EFFECT OF A TRANSMEMBRANE AMINO-ACID ON ETOMIDATE SENSITIVITY OF AN INVERTEBRATE GABA RECEPTOR

1 The gamma-aminobutyric acid (GABA)-modulatory and GABA-mimetic actio ns of etomidate at mammalian GABA(A) receptors are favoured by beta(2) - or beta(3)- versus beta(1)-subunit containing receptors, a selectivi ty which resides with a single transmembrane amino acid (beta(2 N290), beta(3 N289), beta(1 S290)) Here, we have utilized the Xenopus laevis oocyte expression system in conjunction with the two-point voltage cl amp 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. 2 Complem entary RNA-injected oocytes expressing the wild type Rdl GABA receptor and voltage-clamped at -60 mV responded to bath applied GABA with a c oncentration-dependent inward current response and a calculated EC50 f or GABA of 20 +/- 0.4 mu M. Receptors in which the transmembrane methi onine residue (M314) had been exchanged for an asparagine (Rdl(M314N)) or a serine (Rdl(M314S)) also exhibited a concentration-dependent inw ard current response to GABA, but in both cases with a reduced EC50 Of 4.8 +/- 0.2 mu M. 3 Utilizing the appropriate GABA EC10, etomidate (3 00 mu M) had little effect on the agonist-evoked current of the wild t ype Rdl receptor. By contrast, at Rdl(M314N) receptors, etomidate prod uced a clear concentration-dependent enhancement of GABA-evoked curren ts with a calculated EC50 of 64 +/- 3 mu M and an E-max of 68 +/- 2% ( of the maximum response to GABA). 4 The actions of etomidate at Rdl(M3 14N) receptors exhibited an enantioselectivity common to that found fo r mammalian receptors, with 100 mu M R-(+)-etomidate and S-(-)-etomida te enhancing the current induced by GABA (EC10) to 52 +/- 6% and 12 +/ - 1% of the GABA maximum respectively. 5 The effects of this mutation were selective for etomidate as the GABA-modulatory actions of 1 mM pe ntobarbitone at wild type Rdl (49 +/- 4% of the GABA maximum) and Rdl( M314N) receptors (53 +/- 2% of the GABA maximum) were similar. Additio nally, the modest potentiation of GABA produced by the anaesthetic neu rosteroid 5 alpha-pregnan-3 alpha-ol-20-one (Rdl = 25 +/- 4% of the GA BA maximum) was not altered by this mutation (Rdl(M314N) = 18 +/- 3% O f the GABA maximum). 6 Etomidate acting at beta(1) (S290)-containing m ammalian GABA(A) receptors is known to produce only a modest GABA-modu latory effect. Similarly, etomidate acting at Rdl(M314S) receptors pro duced an enhancement of GABA but the magnitude of the effect was reduc ed compared to Rdl(M314N) receptors. 7 Etomidate acting at human alpha (6) beta(3) gamma(2L) receptors is known to produce a large enhancemen t of GABA-evoked currents and at higher concentrations this anaestheti c directly activates the GABA(A) receptor complex. Mutation of the hum an beta(3) subunit asparagine to methionine (beta(3 N289M) found in th e equivalent position in Rdl completely inhibited both the GABA-modula tory and GABA-mimetic action of etomidate (10-300 mu M) acting at alph a(6) beta(3 N289M gamma 2L) receptors. 8 It was concluded that, althou gh invertebrate and mammalian proteins exhibit limited sequence homolo gy, allosteric modification of their function by etomidate can be infl uenced in a complementary manner by a single amino acid substitution. The results are discussed in relation to whether this amino acid contr ibutes to the anaesthetic binding site, or is essential for transducti on. Furthermore, this study provides a clear example of the specificit y of anaesthetic action.