Distinct structural changes in the GABAA receptor elicited by pentobarbital and GABA.
Distinct structural changes in the GABAA receptor elicited by pentobarbital and GABA.
复制标题
戊巴比妥和 GABA 引起 GABAA 受体的明显结构变化。
DOI：
10.1016/j.bpj.2008.09.037
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发表时间：
2009
影响因子：
3.4
通讯作者：
Jackson,MeyerB
中科院分区：
文献类型：
--
作者：
Muroi,Yukiko;Theusch,CassandraM;Czajkowski,Cynthia;Jackson,MeyerB
巴比妥酸盐戊巴比妥与γ-氨基丁酸A型（GABAA）受体结合，这种相互作用在该药物的麻醉作用中起重要作用。根据浓度的不同，戊巴比妥可以增强（100 -100μM）、激活（100-800μM）或阻断（101 -10 mM）通道，但这三种不同作用的机制尚不清楚。为了研究药物诱导的GABAA受体的结构重排，我们用巯基反应性、环境敏感的荧光探针四甲基罗丹明-6-马来酰亚胺（TMRM）标记了在异种卵母细胞中表达的半胱氨酸突变体受体。然后，我们使用联合电压钳和荧光法监测戊巴比妥诱导的通道活动和局部蛋白运动同时在真实的时间。高浓度的戊巴比妥诱导与细胞外结构域中的两个残基（α 1 L127 C和β 2L 125 C）相连的标记物的TMRM荧光（FTMRM）降低，这两个残基先前已被证明会增加对GABA的FTMRM最小响应。M2跨膜螺旋细胞外末端β 2K 274 C处的标记报告了在应用低调节浓度戊巴比妥期间的小但显著的FTMR增加，并且在较高浓度下显示出更大的FTMR增加。与此相反，GABA减少FTMR在这个网站。这些结果表明，GABA和戊巴比妥诱导不同的结构重排的受体，从而激活受体的不同机制。α 1 L127 C和β 2K 274 C标记物在戊巴比妥阻断通道期间大量改变其荧光。与此相反，印防己毒素阻断剂在这些位点不引起FTMR M的变化，拮抗剂SR95531引起的FTMR M信号模式与其他拮抗剂不同。因此，无论是通过拮抗剂阻断通道还是通过激动剂激活通道，GABAA受体蛋白的结构变化在功能等同的转换过程中是不同的。
The barbiturate pentobarbital binds toγ-aminobutyric acid type A (GABAA) receptors, and this interaction plays an important role in the anesthetic action of this drug. Depending on its concentration, pentobarbital can potentiate (∼10–100μM), activate (∼100–800μM), or block (∼1–10 mM) the channel, but the mechanisms underlying these three distinct actions are poorly understood. To investigate the drug-induced structural rearrangements in the GABAAreceptor, we labeled cysteine mutant receptors expressed inXenopusoocytes with the sulfhydryl-reactive, environmentally sensitive fluorescent probe tetramethylrhodamine-6-maleimide (TMRM). We then used combined voltage clamp and fluorometry to monitor pentobarbital-induced channel activity and local protein movements simultaneously in real time. High concentrations of pentobarbital induced a decrease in TMRM fluorescence (FTMRM) of labels tethered to two residues in the extracellular domain (α1L127C andβ2L125C) that have been shown previously to produce an increase inFTMRMin response to GABA. Label atβ2K274C in the extracellular end of the M2 transmembrane helix reported a small but significantFTMRMincrease during application of low modulating pentobarbital concentrations, and it showed a much greaterFTMRMincrease at higher concentrations. In contrast, GABA decreasedFTMRMat this site. These results indicate that GABA and pentobarbital induce different structural rearrangements in the receptor, and thus activate the receptor by different mechanisms. Labels atα1L127C andβ2K274C change their fluorescence by substantial amounts during channel blockade by pentobarbital. In contrast, picrotoxin blockade produces no change inFTMRMat these sites, and the pattern ofFTMRMsignals elicited by the antagonist SR95531 differs from that produced by other antagonists. Thus, with either channel block by antagonists or activation by agonists, the structural changes in the GABAAreceptor protein differ during transitions that are functionally equivalent.