Johansen T H, Chaudhary A, Verdoorn T A
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6600, USA.
Mol Pharmacol. 1995 Nov;48(5):946-55.
We examined the actions of cyclothiazide, aniracetam, and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI-52466) on recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate receptors. Receptors expressed in Xenopus oocytes or human embryonic kidney 293 cells were characterized using voltage and patch-clamp electrophysiology. Aniracetam and cyclothiazide potentiated AMPA receptor currents by slowing or blocking desensitization. Cyclothiazide was more potent at receptors consisting of flip subunits compared with receptors consisting of flop subunits, whereas aniracetam appeared to be more efficacious at flop receptors. The potency of GYKI-52466 did not differ in heteromeric flip or flop containing AMPA receptors, but GYKI-52466 was less potent at homomeric GluRAi and GluRDi receptors. At heteromeric AMPA receptors, 50 microM cyclothiazide increased the IC50 value for GYKI-52466 significantly. The increase was largest in GluRBi/Di receptors where the IC50 value shifted from 21.9 microM (95% confidence interval, 12.0-39.8 microM) to 126 microM (95% confidence interval, 72.4-214 microM) in the presence of cyclothiazide. In contrast, 100 microM GYKI-52466 did not alter the EC50 of cyclothiazide at GluRBi/Di receptors nor did it markedly change the maximal potentiation induced by cyclothiazide. At GluRBi/Di receptors transiently expressed in human embryonic kidney 293 cells, 30 microM GYKI-52466 inhibited the steady state and the peak current evoked by 300 microns L-glutamate to the same extent (34.5 +/- 12% and 27.3 +/- 13.0%, respectively; five experiments), 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 microM cyclothiazide, but GYKI-52466 never restored the desensitization that was blocked by cyclothiazide. Furthermore, GYKI-52466 inhibited L-glutamate currents mediated by homomeric Glu6 receptors, which are not potentiated by 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 modulatory effect of GYKI-52466 result from binding to separate sites on recombinant subunits.
我们研究了环噻嗪、阿尼西坦和1-(4-氨基苯基)-4-甲基-7,8-亚甲基二氧基-5H-2,3-苯并二氮杂䓬（GYKI-52466）对重组α-氨基-3-羟基-5-甲基-4-异恶唑丙酸（AMPA）和海人藻酸受体的作用。利用电压钳和膜片钳电生理学技术对非洲爪蟾卵母细胞或人胚肾293细胞中表达的受体进行了表征。阿尼西坦和环噻嗪通过减缓或阻断脱敏作用增强了AMPA受体电流。与由flop亚基组成的受体相比，环噻嗪对由flip亚基组成的受体作用更强，而阿尼西坦似乎对flop受体更有效。GYKI-52466在含有AMPA受体的异源flip或flop中效力无差异，但在同源GluRAi和GluRDi受体中效力较低。在异源AMPA受体中，50μM环噻嗪显著增加了GYKI-52466的IC50值。在环噻嗪存在下，GluRBi/Di受体中的增加最大，IC50值从21.9μM（95%置信区间，12.0 - 39.8μM）变为126μM（95%置信区间，72.4 - 214μM）。相反，100μM GYKI-52466在GluRBi/Di受体处未改变环噻嗪的EC50，也未显著改变环噻嗪诱导的最大增强作用。在人胚肾293细胞中瞬时表达的GluRBi/Di受体处，30μM GYKI-52466对300μM L-谷氨酸诱发的稳态电流和峰值电流的抑制程度相同（分别为34.5±12%和27.3±13.0%；五个实验），且GYKI-52466未改变脱敏的表观速率（在不存在和存在GYKI-52466时，τ分别为15.7±4.7和17.5±8.3毫秒；五个实验）。在存在和不存在10μM环噻嗪的情况下，GYKI-52466均抑制L-谷氨酸电流，但GYKI-52466从未恢复被环噻嗪阻断的脱敏作用。此外，GYKI-52466抑制由同源Glu6受体介导的L-谷氨酸电流，而环噻嗪对其无增强作用。我们的数据表明，环噻嗪对GYKI-52466与其结合位点亲和力的影响是变构的，且环噻嗪的正性调节作用和GYKI-52466的负性调节作用是由于它们与重组亚基上不同的位点结合所致。
