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Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New Background Naphthalene-etomidate, an etomidate analog containing a bulky phenyl ring substituent group, possesses very low γ-aminobutyric acid type A (GABAA) receptor efficacy and acts as an anesthetic-selective competitive antagonist. Using etomidate analogs containing phenyl ring substituents groups that range in volume, we tested the hypothesis that this unusual pharmacology is caused by steric hindrance that reduces binding to the receptor’s open state. Methods The positive modulatory potencies and efficacies of etomidate and phenyl ring–substituted etomidate analogs were electrophysiology defined in oocyte-expressed α1β3γ2L GABAA receptors. Their binding affinities to the GABAA receptor’s two classes of transmembrane anesthetic binding sites were assessed from their abilities to inhibit receptor labeling by the site-selective photolabels 3[H]azi-etomidate and tritiated R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl) barbituric acid. Results The positive modulatory activities of etomidate and phenyl ring–substituted etomidate analogs progressively decreased with substituent group volume, reflecting significant decreases in both potency (P = 0.005) and efficacy (P < 0.0001). Affinity for the GABAA receptor’s two β+ − α– anesthetic binding sites similarly decreased with substituent group volume (P = 0.003), whereas affinity for the receptor’s α+ – β–/γ+ – β– sites did not (P = 0.804). Introduction of the N265M mutation, which is located at the β+ − α– binding sites and renders GABAA receptors etomidate-insensitive, completely abolished positive modulation by naphthalene-etomidate. Conclusions Steric hindrance selectively reduces phenyl ring–substituted etomidate analog binding affinity to the two β+ − α– anesthetic binding sites on the GABAA receptor’s open state, suggesting that the binding pocket where etomidate’s phenyl ring lies becomes smaller as the receptor isomerizes from closed to open.

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Background-Many sedative-hypnotic agents are thought to act by positively modulating γaminobutyric acid type A (GABA A ) receptors. However, for many agents, the location(s) of the binding site(s) responsible for such receptor modulation is uncertain. We previously developed a low efficacy ligand (naphthalene-etomidate) that binds within a homologous set of hydrophobic cavities located at GABA A receptor subunit interfaces in the transmembrane domain and thus acts as a competitive antagonist for higher efficacy sedative-hypnotics that also bind to these sites. In this report, we describe studies using this compound as a pharmacological screening tool to test whether sedative-hypnotics representing a range of chemical classes can modulate GABA A receptors by binding within these receptor cavities.Methods-The impact of naphthalene-etomidate on GABA-evoked currents that were mediated by oocyte-expressed α 1 β 3 γ 2L GABA A receptors and potentiated by muscimol, alphaxalone, 2,2,2trichloroethanol, isoflurane, AA29504, loreclezole, or diazepam was quantified using electrophysiological techniques.Results-Naphthalene-etomidate (300 μM) significantly reduced GABA A receptor currents potentiated by alphaxalone (by 22 ± 11%), 2,2,2-trichloroethanol (by 23 ± 6%), isoflurane (by 32 ± 10%), AA29504 (by 41 ± 6%), loreclezole (by 43 ± 9%), but significantly increased those potentiated by muscimol (by 26 ± 11%). Naphthalene-etomidate significantly increased currents potentiated by a low (1 μM) diazepam concentration (by 56 ± 14%) while reducing those potentiated by a high (100 μM) diazepam concentration (by 11 ± 7%).Terms of use and reuse: academic research for non-commercial purposes, see here for full terms. https://www.springer.com/aamterms-v1