A high-throughput assay utilizing the voltage/ion probe reader (VIPR) technology identified salicylidene salicylhydrazide (SCS) as being a potent selective inhibitor of α2β1γ1θ GABAA receptors with a maximum inhibition of 56±5% and an IC50 of 32 (23, 45) nM.
Evaluation of this compound using patch-clamp electrophysiological techniques demonstrated that the compound behaved in a manner selective for receptors containing the β1 subunit (e.g. maximum inhibition of 68.1±2.7% and IC50 value of 5.3 (4.4, 6.5) nM on α2β1γ1 receptors). The presence of a β1 subunit was paramount for the inhibition with changes between α1 and α2, γ1 and γ2, and the presence of a θ subunit having little effect.
On all subtypes, SCS produced incomplete inhibition with the greatest level of inhibition at α1β1γ1θ receptors (74.3±1.4%). SCS displayed no use or voltage dependence, suggesting that it does not bind within the channel region. Concentration – response curves to GABA in the presence of SCS revealed a reduction in the maximum response with no change in the EC50 or Hill coefficient. In addition, SCS inhibited pentobarbitone-induced currents.
Threonine 255, located within transmembrane domain (TM) 1, and isoleucine 308, located extracellularly just prior to TM3, were required for inhibition by SCS.
SCS did not compete with the known allosteric modulators, picrotoxin, pregnenolone sulphate, dehydroepiandrosterone 3-sulphate, bicuculline, loreclezole or mefenamic acid. Neither was the inhibition by SCS influenced by the benzodiazepine site antagonist flumazenil.
In conclusion, SCS is unique in selectively inhibiting GABAA receptors containing the β1 subunit via an allosteric mechanism. The importance of threonine 255 and isoleucine 308 within the β1 subunit and the lack of interaction with a range of GABAA receptor modulators suggests that SCS is interacting at a previously unidentified site.
British Journal of Pharmacology (2004) 142, 97–106. doi:10.1038/sj.bjp.0705689