Gurley D, Amin J, Ross P C, Weiss D S, White G
Neurogen Corporation, Branford, CT 06405, USA.
Recept Channels. 1995;3(1):13-20.
Site-directed mutagenesis and the two-electrode voltage-clamp techniques were used to evaluate the site of action of picrotoxin on rat alpha 1 beta 2 gamma 2 containing GABAA receptors expressed in Xenopus oocytes. Following a sequence comparison between GABAA subunits and the picrotoxin-insensitive glycine beta subunit, the following mutations were made near the center of the M2 region of the alpha 1, beta 2, and gamma 2 GABAA subunits: alpha 1(T261F/T267A), beta 2(T246F/T252A), and gamma 2(T271F/T277A). Wild type (alpha 1 beta 2 gamma 2) GABA channels had an IC50 for picrotoxin of 1.3 +/- O.3 microM. In contrast, alpha 1 beta 2 gamma 2 channels that contained any one of the mutated alpha 1, beta 2, or gamma 2 subunits produced currents that were insensitive to picrotoxin (0.1-100 microM). The single mutant beta 2(T246F), in combination with wild type alpha and gamma subunits, also conferred picrotoxin-insensitivity. In contrast, combinations containing beta 2(T252A) were blocked by picrotoxin with an IC50 of 1.4 +/- 0.4 microM. In some instances, the EC50 to GABA was slightly altered in the mutant receptors; but no change was observed in EC50 or potentiation by the allosteric modulator, alprazolam. The data in this study suggest that picrotoxin's site of action is within the channel pore; however the mechanism by which picrotoxin blocks current remains unknown.
采用定点诱变和双电极电压钳技术，评估苦味毒素对非洲爪蟾卵母细胞中表达的含大鼠α1β2γ2的GABAA受体的作用位点。在对GABAA亚基和对苦味毒素不敏感的甘氨酸β亚基进行序列比较后，在α1、β2和γ2 GABAA亚基的M2区域中心附近进行了以下突变：α1（T261F/T267A）、β2（T246F/T252A）和γ2（T271F/T277A）。野生型（α1β2γ2）GABA通道对苦味毒素的IC50为1.3±0.3微摩尔。相比之下，含有突变的α1、β2或γ2亚基之一的α1β2γ2通道产生的电流对苦味毒素（0.1 - 100微摩尔）不敏感。单个突变体β2（T246F）与野生型α和γ亚基组合时，也表现出对苦味毒素不敏感。相反，含有β2（T252A）的组合被苦味毒素阻断，IC50为1.4±0.4微摩尔。在某些情况下，突变受体对GABA的EC50略有改变；但未观察到EC50或变构调节剂阿普唑仑的增强作用有变化。本研究中的数据表明，苦味毒素的作用位点在通道孔内；然而，苦味毒素阻断电流的机制仍不清楚。
