{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T20:00:48Z","timestamp":1789675248048,"version":"4.0.1"},"reference-count":33,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2021,12,21]],"date-time":"2021-12-21T00:00:00Z","timestamp":1640044800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Benzodiazepines (BZDs) produce versatile pharmacological actions through positive modulation of GABAA receptors (GABAARs). A previous study has demonstrated that high concentrations of diazepam potentiate GABA currents on the \u03b11\u03b22\u03b32 and \u03b11\u03b22 GABAARs in a flumazenil-insensitive manner. In this study, the high-concentration effects of BZDs and their sensitivity to flumazenil were determined on synaptic (\u03b11\u03b22\u03b32, \u03b12\u03b22\u03b32, \u03b15\u03b22\u03b32) and extra-synaptic (\u03b14\u03b22\u03b4) GABAARs using the voltage-clamp electrophysiology technique. The in vivo evaluation of flumazenil-insensitive BZD effects was conducted in mice via the loss of righting reflex (LORR) test. Diazepam induced biphasic potentiation on the \u03b11\u03b22\u03b32, \u03b12\u03b22\u03b32 and \u03b15\u03b22\u03b32 GABAARs, but did not affect the \u03b14\u03b22\u03b4 receptor. In contrast to the nanomolar component of potentiation, the second potentiation elicited by micromolar diazepam was insensitive to flumazenil. Midazolam, clonazepam, and lorazepam at 200 \u00b5M exhibited similar flumazenil-insensitive effects on the \u03b11\u03b22\u03b32, \u03b12\u03b22\u03b32 and \u03b15\u03b22\u03b32 receptors, whereas the potentiation induced by 200 \u00b5M zolpidem or triazolam was abolished by flumazenil. Both the GABAAR antagonist pentylenetetrazol and Fa173, a proposed transmembrane site antagonist, abolished the potentiation induced by 200 \u00b5M diazepam. Consistent with the in vitro results, flumazenil antagonized the zolpidem-induced LORR, but not that induced by diazepam or midazolam. Pentylenetetrazol and Fa173 antagonized the diazepam-induced LORR. These findings support the existence of non-classical BZD binding sites on certain GABAAR subtypes and indicate that the flumazenil-insensitive effects depend on the chemical structures of BZD ligands.<\/jats:p>","DOI":"10.3390\/ijms23010042","type":"journal-article","created":{"date-parts":[[2021,12,22]],"date-time":"2021-12-22T01:08:09Z","timestamp":1640135289000},"page":"42","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["High-Dose Benzodiazepines Positively Modulate GABAA Receptors via a Flumazenil-Insensitive Mechanism"],"prefix":"10.3390","volume":"23","author":[{"given":"Na","family":"Wang","sequence":"first","affiliation":[{"name":"State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jingjing","family":"Lian","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanqing","family":"Cao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alai","family":"Muheyati","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shanshan","family":"Yuan","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yujie","family":"Ma","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuzhuo","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gang","family":"Yu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruibin","family":"Su","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Key Laboratory of Neuropsychopharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,12,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"795","DOI":"10.2174\/1568026023393507","article-title":"Subunit composition, distribution and function of GABA(A) receptor subtypes","volume":"2","author":"Sieghart","year":"2002","journal-title":"Curr. 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