Dillon G H, Im H K, Hamilton B J, Carter D B, Gammill R B, Judge T M, Im W B
Upjohn Company, Kalamazoo, Michigan 49001.
Mol Pharmacol. 1993 Oct;44(4):860-5.
We discovered the ability of U-93631 (4-dimethyl-3-t-butylcarboxyl-4,5- dihydro[1,5-a]imidazoquinoxaline) to accelerate decay of gamma-aminobutyric acid (GABA)-induced currents, and we explored its mechanism in human embryonic kidney cells (HEK-293) stably expressing the alpha 1 beta 2 gamma 2 subtype of GABAA receptors. Inward currents (Cl- efflux) induced by 5 microM GABA at the holding potential of -60 mV (under a symmetrical Cl- gradient) decayed with an exponential time course with a mean time constant (tau) of 222 +/- 25 sec, as examined with the whole-cell configuration of the patch-clamp technique. The monoexponential decay was greatly accelerated in the presence of U-93631 at 5 microM, with the mean tau value being 5.2 +/- 0.5 sec. The tau values were dependent on the concentration of U-93631, with an estimated Kd of approximately 2 microM. Outward currents at the holding potential of +60 mV decayed with a similar tau value in the presence of the drug, suggesting the voltage independence of the drug action. The initial amplitude of the GABA (5 microM)-induced Cl- current was not affected by preincubation with U-93631 (5 microM) or GABA (200 nM) alone but was reduced by preincubation with the combination of the two. In the presence of U-93631 at 5 microM, the peak amplitude decreased as a function of GABA concentration, with the half-maximal inhibitory concentration being approximately 100 nm, which is close to the Kd for the high affinity GABA site (85 nM). It appears that the drug interacts with GABA-bound receptors (at least monoliganded) and accelerates receptor desensitization, rather than acting as an open channel blocker. The binding site for U-93631 on GABAA receptors seems not to overlap with GABA, barbiturate, or benzodiazepine sites, because the drug effect persisted in the presence of excess ligands for those sites. With cloned GABAA receptors composed of only alpha 1 beta 2, beta 2 gamma 2, or alpha 1 gamma 2 subunits, U-93631 also accelerated the decay rate. This lack of subtype selectivity raises the possibility that the compound interacts with a region common among the three subunits, probably a novel modulatory site, which can possibly be exploited as a novel therapeutic target.
我们发现U-93631（4-二甲基-3-叔丁基羧基-4,5-二氢[1,5-a]咪唑并喹喔啉）具有加速γ-氨基丁酸（GABA）诱导电流衰减的能力，并在稳定表达GABAA受体α1β2γ2亚型的人胚肾细胞（HEK-293）中探究了其作用机制。在-60 mV的钳制电位下（在对称的Cl-梯度下），用膜片钳技术的全细胞模式检测发现，5 μM GABA诱导的内向电流（Cl-外流）以指数时间进程衰减，平均时间常数（τ）为222±25秒。在存在5 μM U-93631的情况下，单指数衰减大大加速，平均τ值为5.2±0.5秒。τ值取决于U-93631的浓度，估计解离常数（Kd）约为2 μM。在药物存在下，+60 mV钳制电位下的外向电流以相似的τ值衰减，表明药物作用与电压无关。GABA（5 μM）诱导的Cl-电流的初始幅度不受单独用U-93631（5 μM）或GABA（200 nM）预孵育的影响，但受两者组合预孵育的影响而降低。在存在5 μM U-93631的情况下，峰值幅度随GABA浓度的增加而降低，半数最大抑制浓度约为100 nM，这与高亲和力GABA位点的Kd（85 nM）接近。看来该药物与结合了GABA的受体（至少是单配体的）相互作用并加速受体脱敏，而不是作为开放通道阻滞剂起作用。U-93631在GABAA受体上的结合位点似乎不与GABA、巴比妥酸盐或苯二氮䓬位点重叠，因为在存在这些位点的过量配体时药物效应仍然存在。对于仅由α1β2、β2γ2或α1γ2亚基组成的克隆GABAA受体，U-93631也加速了衰减速率。这种缺乏亚型选择性增加了该化合物与这三个亚基共有的区域相互作用的可能性，可能是一个新的调节位点，有可能被开发为新的治疗靶点。
