INTERACTIONS AMONG GYKI-52466, CYCLOTHIAZIDE, AND ANIRACETAM AT RECOMBINANT AMPA AND KAINATE RECEPTORS

We examined the actions of cyclothiazide, aniracetam, and -4-methyl-7, 8-methylenedioxy-5H-2,3-benzodiazepine (GYKI-52466) on recombinant alp ha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate receptors. Receptors expressed in Xenopus oocytes or human embryonic k idney 293 cells were characterized using voltage and patch-clamp elect rophysiology. Aniracetam and cyclothiazide potentiated AMPA receptor c urrents by slowing or blocking desensitization. Cyclothiazide was more potent at receptors consisting of flip subunits compared with recepto rs consisting of flop subunits, whereas aniracetam appeared to be more efficacious at flop receptors. The potency of GYKI-52466 did not diff er in heteromeric flip or flop containing AMPA receptors, but GYKI-524 66 was less potent at homomeric GluRA(i) and GluRD(i) receptors. At he teromeric AMPA receptors, 50 mu M cyclothiazide increased the IC50 val ue for GYKI-52466 significantly. The increase was largest in GluRB(i)/ D-i receptors where the IC50 value shifted from 21.9 mu M (95% confide nce interval, 12.0-39.8 mu M) to 126 mu M (95% confidence interval, 72 .4-214 mu M) in the presence of cyclothiazide. In contrast, 100 mu M G YKI-52466 did not alter the EC(50) of cyclothiazide at GluRB(i)/D-i re ceptors nor did it markedly change the maximal potentiation induced by cyclothiazide. At GluRB(i)/D-i receptors transiently expressed in hum an embryonic kidney 293 cells, 30 mu M GYKI-52466 inhibited the steady state and the peak current evoked by 300 mu m L-glutamate to the same extent (34.5 +/- 12% and 27.3 +/- 13.0%, respectively; five experimen ts), and GYKI-52466 did not alter the apparent rate of desensitization (tau = 15.7 +/- 4.7 and 17.5 +/- 8.3 msec in the absence and presence of GYKI-52466, respectively; five experiments). GYKI-52466 inhibited L-glutamate currents in the presence and absence of 10 mu M cyclothiaz ide, but GYKI-52466 never restored the desensitization that was blocke d by cyclothiazide. Furthermore, GYKI-52466 inhibited L-glutamate curr ents mediated by homomeric Glu, receptors, which are not potentiated b y cyclothiazide. Our data suggest that the effect of cyclothiazide on the affinity of GYKI-52466 for its binding site is allosteric and that the positive modulatory effect of cyclothiazide and the negative modu latory effect of GYKI-52466 result from binding to separate sites on r ecombinant subunits.