The neurosteroid 5ß-pregnan-3α-ol-20-one enhances actions of etomidate as a positive allosteric modulator of α1ß2γ2L GABAA receptors.

BACKGROUND AND PURPOSE: Neurosteroids potentiate responses of the GABAA receptor to the endogenous agonist GABA. Here, we examined the ability of neurosteroids to potentiate responses to the allosteric activators etomidate, pentobarbital and propofol. EXPERIMENTAL APPROACH: Electrophysiological assays were conducted on rat α1ß2γ2L GABAA receptors expressed in HEK 293 cells. The sedative activity of etomidate was studied in Xenopus tadpoles and mice. Effects of neurosteroids on etomidate-elicited inhibition of cortisol synthesis were determined in human adrenocortical cells. KEY RESULTS: The neurosteroid 5ß-pregnan-3α-ol-20-one (3α5ßP) potentiated activation of GABAA receptors by GABA and allosteric activators. Co-application of 1 µM 3α5ßP induced a leftward shift (almost 100-fold) of the whole-cell macroscopic concentration-response relationship for gating by etomidate. Co-application of 100 nM 3α5ßP reduced the EC50 for potentiation by etomidate of currents elicited by 0.5 µM GABA by about three-fold. In vivo, 3α5ßP (1mg kg(-1) ) reduced the dose of etomidate required to produce loss of righting in mice (ED50 ) by almost 10-fold. In tadpoles, the presence of 50 or 100 nM 3α5ßP shifted the EC50 for loss of righting about three- or ten-fold respectively. Exposure to 3α5ßP did not influence inhibition of cortisol synthesis by etomidate. CONCLUSIONS AND IMPLICATIONS: Potentiating neurosteroids act similarly on orthosterically and allosterically activated GABAA receptors. Co-application of neurosteroids with etomidate can significantly reduce dosage requirements for the anaesthetic, and is a potentially beneficial combination to reduce undesired side effects.

Mechanisms of potentiation of the mammalian GABAA receptor by the marine cembranoid eupalmerin acetate.

BACKGROUND AND PURPOSE: Eupalmerin acetate (EPA) is a marine diterpene compound isolated from the gorgonian octocorals Eunicea succinea and Eunicea mammosa. The compound has been previously shown to modulate muscle-type and neuronal nicotinic acetylcholine receptors, which are inhibited in the presence of low micromolar concentrations of EPA. In this study, we examined the effect of EPA on another transmitter-gated ion channel, the GABA(A) receptor. EXPERIMENTAL APPROACH: Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat wild-type and mutant alpha1beta2gamma2L GABA(A) receptors. KEY RESULTS: Our findings demonstrate that, at micromolar concentrations, EPA potentiates the rat alpha1beta2gamma2L GABA(A) receptor. The analysis of single-channel currents recorded in the presence of EPA showed that the kinetic mode of action of EPA is similar to that of neuroactive steroids. Mutations to residues alpha1Q241 and alpha1N407/Y410, previously shown to affect receptor modulation by neurosteroids, also diminished potentiation by EPA. Exposure to a steroid antagonist, (3alpha,5alpha)-17-phenylandrost-16-en-3-ol, reduced potentiation by EPA. Additionally, exposure to EPA led to potentiation of GABA(A) receptors activated by very high concentrations (1-10 microM) of allopregnanolone. In tadpole behavioural assays, EPA caused loss of righting reflex and loss of swimming reflex. CONCLUSIONS AND IMPLICATIONS: We conclude that EPA either interacts with the putative neurosteroid binding site on the GABA(A) receptor or shares with neurosteroids the key transduction elements involved in channel potentiation by steroids. The results indicate that cembranoids represent a novel class of GABA(A) receptor modulators.