Competitive Interactions Between Propofol and Diazepam: Studies in GABA_A Receptors and Zebrafish

Although propofol is among the most commonly administered general anesthetics, its mechanism of action is not fully understood. It has been hypothesized that propofol acts via a similar mechanism as (R)-ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (etomidate) by binding within the GABA_A receptor transmembrane receptor domain at the two β ⁺/α ^- subunit interfaces with resultant positive allosteric modulation. To test this hypothesis, we leveraged the ability of diazepam to bind to those sites and act as a competitive antagonist. We used oocyte-expressed α ₁ β ₃ γ _2L GABA_A receptors to define the actions of diazepam (± flumazenil) on currents activated or potentiated by propofol and a zebrafish activity assay to define the impact of diazepam and flumazenil on propofol-induced anesthesia. We found that diazepam increased the amplitudes of GABA_A receptor-mediated currents at nanomolar concentrations but reduced them at micromolar concentrations. The current amplitude changes produced by nanomolar diazepam concentrations were inhibited by flumazenil whereas those produced by micromolar diazepam concentrations were not. Studies of agonist potentiation showed that the micromolar inhibitory action of diazepam was surmountable by high concentrations of propofol and produced a rightward shift in the propofol concentration-response curve characterized by a Schild slope not statistically significantly different from 1, consistent with competition between diazepam and propofol. Although micromolar concentrations of diazepam (plus flumazenil) similarly reduced GABA_A receptor currents modulated by propofol and etomidate, it only reduced the anesthetic actions of etomidate. We conclude that while both propofol and etomidate can modulate GABA_A receptors by binding to the β ⁺/α ^- subunit interfacial sites, propofol-induced anesthesia likely involves additional target sites. SIGNIFICANCE STATEMENT: Although the drug combination of diazepam and flumazenil reverses the GABA_A receptor positive modulatory actions of both propofol and (R)-ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (etomidate), it only reverses the in vivo anesthetic actions of etomidate. These results strongly suggest that distinct mechanisms of action account for the anesthetic actions of these two commonly administered anesthetic agents.