Propofol is a widely used anesthetic and sedative that acts as a positive allosteric modulator of gamma-aminobutyric acid type A (GABAA) receptors. Several potential propofol binding sites that may mediate this effect have been identified using propofol-analogue photoaffinity labeling. Ortho-propofol diazirine (o-PD) labels β-H267, a pore-lining residue, whereas AziPm labels residues β-M286, β-M227, and α-I239 in the two membrane-facing interfaces [β(+)/α(−) and α(+)/β(−)] between α and β subunits. This study used photoaffinity labeling of α1β3 GABAA receptors to reconcile the apparently conflicting results obtained with AziPm and o-PD labeling, focusing on whether β3-H267 identifies specific propofol binding site(s). The results show that propofol, but not AziPm protects β3-H267 from labeling by o-PD, whereas both propofol and o-PD protect against AziPm labeling of β3-M286, β3-M227, and α1I239. These data indicate that there are three distinct classes of propofol binding sites, with AziPm binding to two of the classes and o-PD to all three. Analysis of binding stoichiometry using native mass spectrometry in β3 homomeric receptors, demonstrated a minimum of five AziPm labeled residues and three o-PD labeled residues per pentamer, suggesting that there are two distinct propofol binding sites per β-subunit. The native mass spectrometry data, coupled with photolabeling performed in the presence of zinc, indicate that the binding site(s) identified by o-PD are adjacent to, but not within the channel pore, since the pore at the 17′ H267 residue can accommodate only one propofol molecule. These data validate the existence of three classes of specific propofol binding sites on α1β3 GABAA receptors.

中文翻译：

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GABAA 受体上的三类异丙酚结合位点


异丙酚是一种广泛使用的麻醉剂和镇静剂，可作为 A 型 γ-氨基丁酸 （GABAA） 受体的正变构调节剂。已经使用异丙酚类似物光亲和标记确定了几个可能介导这种效应的潜在异丙酚结合位点。邻丙泊酚二氮嗪 （o-PD） 标记 β-H267（一种孔内壁残基），而 AziPm 标记 α 和 β 亚基之间两个面向膜的界面 [β（+）/α（−） 和 α（+）/β（−）] 中的残基 β-M286、β-M227 和 α-I239。本研究使用 α1β3 GABAA 受体的光亲和标记来调和与 AziPm 和 o-PD 标记获得的明显冲突的结果，重点关注 β3-H267 是否识别特定的异丙酚结合位点。结果表明，异丙酚而不是 AziPm 保护 β3-H267 免受 o-PD 标记，而异丙酚和 o-PD 都防止 β3-M286、β3-M227 和 α1I239 的 AziPm 标记。这些数据表明，异丙酚结合位点分为三类，其中 AziPm 与其中两类结合，而 o-PD 与这三类结合。使用 β3 同聚体受体中的天然质谱法对结合化学计量法进行分析，结果表明每个五聚体至少有 5 个 AziPm 标记残基和 3 个 o-PD 标记残基，表明每个 β 亚基有两个不同的异丙酚结合位点。天然质谱数据，加上在锌存在下进行的光标记，表明 o-PD 鉴定的结合位点与通道孔相邻，但不在通道孔内，因为 17′ H267 残基处的孔只能容纳一个异丙酚分子。这些数据验证了 α1β3 GABAA 受体上存在三类特异性异丙酚结合位点。