Competitive Antagonism of Etomidate Action by Diazepam In Vitro GABAA Receptor and In Vivo Zebrafish Studies

Background: Recent cryo-electron microscopic imaging studies have shown that in addition to binding to the classical extracellular benzodiazepine binding site of the alpha(1)beta(3)gamma(2L), gamma-aminobutyric acid type A (GABA(A)) receptor, diazepam also binds to etomidate binding sites located in the transmembrane receptor domain. Because such binding is characterized by low modulatory efficacy, the authors hypothesized that diazepam would act in vitro and in vivo as a competitive etomidate antagonist. Methods: The concentration-dependent actions of diazepam on 20 mu M etomidate-activated and 6 mu M GABA-activated currents were defined (in the absence and presence of flumazenil) in oocyte-expressed alpha(1)beta(3)gamma(2L) GABA(A) receptors using voltage clamp electrophysiology. The ability of diazepam to inhibit receptor labeling of purified alpha(1)beta(3)gamma(2L) GABA(A) receptors by 3[H]azietomidate was assessed in photoaffinity labeling protection studies. The impact of diazepam (in the absence and presence of flumazenil) on the anesthetic potencies of etomidate and ketamine was compared in a zebrafish model. Results: At nanomolar concentrations, diazepam comparably potentiated etomidate-activated and GABA-activated GABA(A) receptor peak current amplitudes in a flumazenil-reversible manner. The half-maximal potentiating concentrations were 39 nM (95% CI, 27 to 55 nM) and 26 nM (95% CI, 16 to 41 nM), respectively. However, at micromolar concentrations, diazepam reduced etomidate-activated, but not GABA-activated, GABA(A) receptor peak current amplitudes in a concentration-dependent manner with a half-maximal inhibitory concentration of 9.6 mu M (95% CI, 7.6 to 12 mu M). Diazepam (12.5 to 50 mu M) also right-shifted the etomidate-concentration response curve for direct activation without reducing the maximal response and inhibited receptor photoaffinity labeling by 3[H]azietomidate. When administered with flumazenil, 50 mu M diazepam shifted the etomidate (but not the ketamine) concentration-response curve for anesthesia rightward, increasing the etomidate EC50 by 18-fold. Conclusions: At micromolar concentrations and in the presence of flumazenil to inhibit allosteric modulation via the classical benzodiazepine binding site of the GABA(A) receptor, diazepam acts as an in vitro and in vivo competitive etomidate antagonist.