1 A high-throughput assay utilizing the voltage/ion probe reader (VIPR) technology identified salicylidene salicylhydrazide (SCS) as being a potent selective inhibitor of a2b1g1y GABA A receptors with a maximum inhibition of 5675% and an IC 50 of 32 (23, 45) nM. 2 Evaluation of this compound using patch-clamp electrophysiological techniques demonstrated that the compound behaved in a manner selective for receptors containing the b1 subunit (e.g. maximum inhibition of 68.172.7% and IC 50 value of 5.3 (4.4, 6.5) nM on a2b1g1 receptors). The presence of a b1 subunit was paramount for the inhibition with changes between a1 and a2, g1 and g2, and the presence of a y subunit having little effect. 3 On all subtypes, SCS produced incomplete inhibition with the greatest level of inhibition at a1b1g1y receptors (74.371.4%). 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 EC 50 or Hill coefficient. In addition, SCS inhibited pentobarbitone-induced currents. 4 Threonine 255, located within transmembrane domain (TM) 1, and isoleucine 308, located extracellularly just prior to TM3, were required for inhibition by SCS. 5 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. 6 In conclusion, SCS is unique in selectively inhibiting GABA A receptors containing the b1 subunit via an allosteric mechanism. The importance of threonine 255 and isoleucine 308 within the b1 subunit and the lack of interaction with a range of GABA A receptor modulators suggests that SCS is interacting at a previously unidentified site.
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