ALLOSTERIC REGULATION OF HA-AMINO-3-HYDROXY-5-METHYL-4-ISOXAZOLE-PROPIONATE RECEPTORS BY THIOCYANATE AND CYCLOTHIAZIDE AT A COMMON MODULATORY SITE DISTINCT FROM THAT OF 2,3-BENZODIAZEPINES

Allosteric regulators of ha-amino-3-hydroxy-5-methyl-4-isoxazole-propi onate (AMPA) receptors include 2,3-benzodiazepines such as GYKI 52466 and GYKI 53655 and the chaotropic anion thiocyanate that inhibit, and benzothiadiazines such as cyclothiazide that potentiate AMPA receptor currents. Here we sought to determine whether the allosteric regulator s modulate AMPA receptors at a common or distinct allosteric sites by comparing their actions on AMPA- and kainate-evoked currents in cultur ed rat hippocampal neurons and Xenopus oocytes expressing recombinant AMPA receptor subunits. GYKI 52466 and thiocyanate blocked AMPA-evoked currents in a concentration-dependent manner (IC50 values, 8.2 mu M a nd 1.1 mM, respectively); in contrast, kainate-evoked currents were bl ocked by GYKI 52466, but were potentiated by high concentrations of th iocyanate (greater than or equal to 3 mM). Thiocyanate enhanced the ra te of desensitization and slowed recovery from desensitization of AMPA -evoked currents. whereas GYKI 52466 failed to affect desensitization. Among neurons in the hippocampal cultures, there was cell-to-cell var iability in the sensitivity to block of AMPA-evoked currents by thiocy anate that was correlated with the degree of potentiation by cyclothia zide. Moreover, cyclothiazide caused a parallel rightward shift in the concentration-response curve for thiocyanate block, and slowed the on set of thiocyanate block to a rate that was similar to that of cycloth iazide dissociation. Together. these observations suggest that thiocya nate and cyclothiazide act at non-distinct allosteric sites. GYKI 5246 6 blocked AMPA receptor responses to a similar extent, irrespective of the degree of cyclothiazide potentiation. Moreover, the kinetics of G YKI 53655 block in the presence of cyclothiazide were not consistent w ith a competitive interaction. As is the case for cyclothiazide, SCN- exhibited greater affinity for Pip than for pop AMPA receptor splice v ariants. In particular, GluR1(flip)/GluR2(flip) was especially sensiti ve to thiocyanate block. We conclude that thiocyanate, a flip-preferri ng allosteric modulator like cyclothiazide, appears to act by enhancin g desensitization at a site that may overlap the site where cyclothiaz ide reduces desensitization, whereas 2.3-benzodiazepines act at a dist inct site and the block does not involve a modification of desensitiza tion.