The interaction of general anaesthetics with recombinant GABAA and glycine receptors expressed in Xenopus laevis oocytes:: a comparative study

1 The effects of five structurally dissimilar general anaesthetics were examined in voltage-clamp recordings of agonist-evoked currents mediated by recombinant gamma-aminobutyric acid (GABA)(A) receptors composed of human alpha(1) beta(1) and gamma(2L) subunits expressed in Xenopus laevis oocytes. A quantitative comparison of the effects of these agents was made upon recombinant glycine receptors expressed as a homo-oligomer of human alpha(1) subunits, or as a hetero-oligomer of human alpha(1) and rat beta subunits. 2 Complementary RNA-injected oocytes expressing GABA(A) receptors responded to bath applied GABA with an EC50 of 158+/-34 mu M. Oocytes expressing alpha(1) and alpha(1) beta glycine receptors subsequent to cDNA injection displayed EC50 values of 76+/-2 mu M and 66+/-2 mu M, respectively, in response to bath applied glycine. 3 Picrotoxin antagonized responses mediated by homo-oligomeric alpha(1) glycine receptors with an IC50 of 4.2+/-0.8 mu M. Hetero-oligomeric alpha(1) beta glycine receptors were at least 100-fold less sensitive to blockade by picrotoxin. 4 With the appropriate agonist EC10, propofol enhanced GABA and glycine-evoked currents to approximately the maximal response produced by a saturating concentration of either agonist (i.e. I-max). The calculated EC50 values were 2.3+/-0.2 mu M, 16+/-3 mu M and 27+/-2 mu M, for GABA(A) alpha(1) beta(1) gamma(2L), glycine alpha(1) and alpha(1) beta receptors, respectively. At relatively high concentrations, propofol was observed to activate directly both GABA(A) glycine receptors. 5 Pentobarbitone potentiated GABA-evoked currents to 117+/-8.5% of I-max, with an EC50 of 65+/-3 mu M. The barbiturate also produced a substantial enhancement of the glycine-evoked currents, I-max and EC50 values being 71+/-2% and 845+/-66 mu M and 51+/-10% and 757+/-30 mu M for homomeric alpha(1) and heteromeric alpha(1) beta glycine receptors respectively. At high concentrations, pentobarbitone directly activated GABA(A), but not glycine, receptors. 6 The potentiation by propofol or pentobarbitone of currents mediated by alpha(1) homo-oligomeric glycine receptors was in both cases associated with a parallel sinistral shift of the glycine concentration-effect curve. The effects of binary combinations of pentobarbitone and propofol at maximally effective concentrations were mutually occlusive suggesting a common site, or mechanism, of action. 7 GABA-evoked currents were maximally potentiated by etomidate to 79+/-2% of I-max (EC50 of 8.1+/-0.9 mu M). By contrast, glycine-induced currents mediated by alpha(1) and alpha(1) beta glycine receptor isoforms were enhanced only to 29+/-4% and 28+/-3% of I-max. Limited solubility precluded the calculation of EC50 values for the effect of etomidate at glycine receptors. None of the receptor isoforms examined were directly activated by etomidate. 8 The neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one potentiated GABA-evoked currents to 69+/-4% of I-max, with an EC50 value of 89+/-6 nM. In contrast, both alpha(1) homo-oligomeric and alpha(1) beta heter-oligomeric glycine receptors were insensitive to the action of this steroid. A direct agonist action of the steroid was discernible at GABA(A), but not glycine, receptors. 9 Trichloroethanol, the active metabolite of the general anaesthetic chloral hydrate, enhanced glycine-evoked currents to 77+/-10% and 94+/-4% of I-max on alpha(1) and alpha(1) beta glycine receptors, with EC50 values of 3.5+/-0.1 mM and 5.9+/-0.3 mM respectively. On GABA(A) receptors, trichloroethanol had a lower maximum enhancement (52+/-5% of I-max), but a slightly higher potency (EC50 1.0+/-0.1 mM). Trichloroethanol activated neither GABA(A), nor glycine, receptors. 10 The data demonstrate a variety of intravenous general anaesthetic agents, at clinically relevant concentrations, to augment preferentially GABA-evoked currents mediated by the alpha(1) beta(1) gamma(2L) receptor subunit combination as compared to their effects on both alpha(1) and alpha(1) beta glycine receptors. However, the presence on glycine receptors of lower affinity modulatory binding sites for pentobarbitone, propofol and trichloroethanol may aid in the identification of the molecular determinants of the CNS actions of these anaesthetics.