Agonist-, antagonist-, and benzodiazepine-induced structural changes in the α1Met113-Leu132 region of the GABAA receptor
Agonist-, antagonist-, and benzodiazepine-induced structural changes in the α1Met113-Leu132 region of the GABAA receptor
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DOI：
10.1124/mol.106.028662
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发表时间：
2007-02-01
影响因子：
3.6
通讯作者：
Czajkowski, Cynthia
中科院分区：
文献类型：
--
作者：
Kloda, Jessica Holden;Czajkowski, Cynthia
激动剂、拮抗剂和变构调节剂对配体门控离子通道发挥其独特作用的结构基础知之甚少。我们使用取代的半胱氨酸可及性方法来探测GABA（A）受体在存在引起不同药理学作用的配体的情况下的结构。GABA结合位点的α（1）Met（113）-Leu（132）区域中的残基单独突变为半胱氨酸，并在非洲爪蟾卵母细胞中与野生型β（2）和γ（2）亚基一起表达。利用电生理学方法，测定了N-生物素氨乙基甲硫基磺酸盐（MTSEA-生物素），在静息的（未配体）状态，并将其与GABA（激动剂），4-[6-亚氨基-3-甲基-N-氨基-N-甲基-N（4-甲氧基苯基）哒嗪-1-基]丁酸氢溴酸盐（SR-95531;拮抗剂）、戊巴比妥（变构调节剂）和泮（变构调节剂）。预测α（1）N115 C、α（1）L117 C、α（1）T129 C和α（1）R131 C与GABA结合口袋对齐，因为这些残基的MTSEA-生物素修饰降低了GABA引发的电流量，并且GABA和SR-95531均降低了修饰的速率/程度。一些取代的半胱氨酸的反应速率是不同的，这取决于配体，表明巴比妥酸盐和GABA诱导的通道门控，拮抗剂结合，和苯二氮卓类调节诱导特定的结构重排。α（1）E122 C的化学反应性被GABA或戊巴比妥降低，但不受SR-95531结合的影响，而α（1）L127 C的反应性被激动剂和拮抗剂结合降低，但不受戊巴比妥的影响。此外，α（1）E122 C、α（1）L127 C和α（1）R131 C在对泮的反应中改变了可及性，提供了GABA结合位点内和附近的残基在对苯二氮卓类调节的反应中移动的结构证据。
The structural basis by which agonists, antagonists, and allosteric modulators exert their distinct actions on ligand-gated ion channels is poorly understood. We used the substituted cysteine accessibility method to probe the structure of the GABA(A) receptor in the presence of ligands that elicit different pharmacological effects. Residues in the alpha(1)Met(113)-Leu(132) region of the GABA binding site were individually mutated to cysteine and expressed with wild-type beta(2) and gamma(2) subunits in Xenopus laevis oocytes. Using electrophysiology, we determined the rates of reaction of N-biotinaminoethyl methanethiosulfonate (MTSEA-biotin) with the introduced cysteines in the resting (unliganded) state and compared them with rates determined in the presence of GABA (agonist), 4-[6-imino-3-(4-methoxyphenyl) pyridazin-1-yl] butanoic acid hydrobromide (SR-95531; antagonist), pentobarbital (allosteric modulator), and flurazepam (allosteric modulator). alpha(1)N115C, alpha(1)L117C, alpha(1)T129C, and alpha(1)R131C are predicted to line the GABA binding pocket because MTSEA-biotin modification of these residues decreased the amount of current elicited by GABA, and the rates/extents of modification were decreased both by GABA and SR-95531. Reaction rates of some substituted cysteines were different depending on the ligand, indicating that barbiturate- and GABA-induced channel gating, antagonist binding, and benzodiazepine modulation induce specific structural rearrangements. Chemical reactivity of alpha(1)E122C was decreased by either GABA or pentobarbital but was unaltered by SR-95531 binding, whereas alpha(1)L127C reactivity was decreased by agonist and antagonist binding but not affected by pentobarbital. Furthermore, alpha(1)E122C, alpha(1)L127C, and alpha(1)R131C changed accessibility in response to flurazepam, providing structural evidence that residues in and near the GABA binding site move in response to benzodiazepine modulation.