Involvement of flumazenil-insensitive benzodiazepine binding site in benzodiazepine-induced anesthesia in zebrafish larvae

OBJECTIVE To identify the involvement of flumazenil-insensitive benzodiazepine(BZD) binding site in mediating BZD-induced immobility.The distribution of this nonclassical binding site and its key amino acid residues in GABAAreceptors(GABAARs) were also investigated.METHODS Using a zebrafish larvae locomotion model,we investigated the detailed dose-dependent effects of diazepam and other BZDs on zebrafish larvae behaviors,with a focus on their high-dose effects.We then evaluated the influence of the classical BZD antagonist flumazenil,GABAARs antagonist bicuculline,and the antagonist of a proposed BZD binding site in α4/6β3δ subtype receptor Ro15-4513 on BZDs induced immobility.Using wholecell patch clamp electrophysiological recordings on recombinant GABAARs,we investigated the modulation of diazepam alone or combined with flumazenil on GABA-elicited current in wildtype and mutated receptors.RESULTS Diazepam dose-dependently decreased the locomotor activities of zebrafish larvae at doses of 0.4,2,10,20,30,50 and 75 mg·L^(-1).The hypolocomotion(sedation-like state) induced by diazepam at10 and 20 mg·L^(-1) were effectively antagonized by flumazenil with EC150 of 0.086 mg·L-and1.295 mg·L^(-1),while the immobility(anesthesialike state) induced by diazepam at 30 mg·L^(-1) was abolished by bicuculline(3 mg·L^(-1)),but not affected by flumazenil(even at concentration up to150 mg·L^(-1)) or Ro15-4513(100 mg·L^(-1)).The immobility induced by clonazepam and lorazepam(100 mg·L^(-1)) was also resistant to flumazenil(100 mg·L^(-1)).In the α1β2γ2 subtype receptor expressed in HEK293 T cells,diazepam dose-dependently potentiated GABA-elicited current,and this potentiation was effectively antagonized by flumazenil(100 μmol·L^(-1)).However,in α1β2 subtype receptor,diazepam(150 μmol·L^(-1)) induced potentiation was insensitive to flumazenil(100 μmol·L^(-1)),but was abolished by the mutation of β2 N265 I.CONCLUSION These results provide direct in vivo evidence for the nonclassical binding sites,which may be located at the second transmembrane domain of GABAAR,mediate BZD-induced anesthesia.