A Cysteine Substitution Probes β3H267 Interactions with Propofol and Other Potent Anesthetics in α1β3γ2L γ-Aminobutyric Acid Type A Receptors.
A Cysteine Substitution Probes β3H267 Interactions with Propofol and Other Potent Anesthetics in α1β3γ2L γ-Aminobutyric Acid Type A Receptors.
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DOI：
10.1097/aln.0000000000000934
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发表时间：
2016-01
期刊：
影响因子：
8.8
通讯作者：
Forman SA
中科院分区：
文献类型：
--
作者：
Stern AT;Forman SA
γ-氨基丁酸 A 型 (GABAA) 受体中的麻醉接触残基已使用光标记进行了鉴定，包括两种异丙酚衍生物。 O-丙泊酚-二氮丙啶标记 β3 和 α1β3 受体中的 H267，而间阿齐-丙泊酚标记 α1β3 亚基间裂隙中的其他残基。这两种标记均未在哺乳动物大脑中最常见的亚型 αβγ 受体中进行过研究。在αβγ受体中，其他麻醉衍生物光标记间阿齐-丙泊酚标记的残基，但βH267没有。我们的 α1β3γ2L 受体结构同源模型表明 β3H267 可能毗邻其中一些位点。使用取代的半胱氨酸修饰保护来测试 β3H267C 与四种强效麻醉剂的相互作用：丙泊酚、依托咪酯、阿法沙酮和 R-5-烯丙基-1-甲基-5-(间三氟甲基-二氮杂苯基苯基)巴比妥酸 (mTFD-MPAB)。我们在非洲爪蟾卵母细胞中表达α1β3γ2L或α1β3H267Cγ2L GABAA受体。我们使用电压钳电生理学来评估受体对 GABA 和麻醉剂的敏感性，并比较 GABA 与 GABA 加麻醉剂对对氯汞苯磺酸盐 (pCMBS) 的修饰率。所有四种麻醉剂的等催眠浓度对 GABA EC5 反应的增强作用在 α1β3γ2L 和 α1β3H267Cγ2L 受体中相似 (n ≥ 3)。 mTFD-MPAB 和异丙酚直接激活 α1β3H267Cγ2L 受体，但不直接激活 α1β3γ2L，显着大于其他麻醉剂。 pCMBS (n ≥ 4) 对 β3H267C 的修饰速度很快，并且 GABA 加速了修饰。仅 mTFD-MPAB 减慢了 β3H267C 修饰（约 2 倍；p = 0.011）。 α1β3γ2L GABAA 受体中的 β3H267 接触 mTFD-MPAB，但不接触异丙酚。我们的结果表明，β3H267 位于 mTFD-MPAB 和丙泊酚结合的一个或两个跨膜亚基间（α+/β− 和 γ+/β−）袋的外围附近。
Anesthetic contact residues in γ-aminobutyric acid type A (GABAA) receptors have been identified using photolabels, including two propofol derivatives. O-propofol-diazirine labels H267 in β3 and α1β3 receptors, while m-azi-propofol labels other residues in intersubunit clefts of α1β3. Neither label has been studied in αβγ receptors, the most common isoform in mammalian brain. In αβγ receptors, other anesthetic derivatives photolabel m-azi-propofol labeled residues, but not βH267. Our structural homology model of α1β3γ2L receptors suggests that β3H267 may abut some of these sites. Substituted cysteine modification-protection was used to test β3H267C interactions with four potent anesthetics: propofol, etomidate, alphaxalone, and R-5-allyl-1-methyl-5-(m-trifluoromethyl-diazirinylphenyl) barbituric acid (mTFD-MPAB). We expressed α1β3γ2L or α1β3H267Cγ2L GABAA receptors in Xenopus oocytes. We used voltage clamp electrophysiology to assess receptor sensitivity to GABA and anesthetics, and to compare para-chloromercuribenzenesulfonate (pCMBS) modification rates with GABA versus GABA plus anesthetics. Enhancement of GABA EC5 responses by equi-hypnotic concentrations of all four anesthetics was similar in α1β3γ2L and α1β3H267Cγ2L receptors (n ≥ 3). Direct activation of α1β3H267Cγ2L receptors, but not α1β3γ2L, by mTFD-MPAB and propofol was significantly greater than the other anesthetics. Modification of β3H267C by pCMBS (n ≥ 4) was rapid and accelerated by GABA. Only mTFD-MPAB slowed β3H267C modification (~2-fold; p = 0.011). β3H267 in α1β3γ2L GABAA receptors contacts mTFD-MPAB, but not propofol. Our results suggest that β3H267 is near the periphery of one or both transmembrane inter-subunit (α+/β− and γ+/β−) pockets where both mTFD-MPAB and propofol bind.