Beecher-Mallinckrodt Laboratories, Department of Anesthesia Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts.
Beecher-Mallinckrodt Laboratories, Department of Anesthesia Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts
Mol Pharmacol. 2023 Dec;104(6):266-274. doi: 10.1124/molpharm.123.000719. Epub 2023 Aug 16.
Multiple approaches, including cryogenic electron microscopy (cryo-EM), indicate that the anesthetics etomidate and propofol modulate 12/32 GABA receptors by binding in overlapping transmembrane inter-subunit sites near M286 and L232 sidechains. High-precision approaches in functional receptors are needed for comparisons with cryo-EM. We previously used substituted cysteine modification and protection (SCAMP) with n-alkyl-methanethiosulfonate (MTS) reagents and electrophysiology in 13M286C2L receptors to estimate the distance from etomidate to 3M286 with precision near 1.3 Å. Here, we address three more aims using this approach: (i) SCAMP with etomidate was tested in 1L232C32L receptors; (ii) studies in 1L232W3M286C2L receptors assessed whether 1L232W displaces etomidate relative to 3M286C; and (iii) results with propofol were compared with those with etomidate. Voltage-clamp electrophysiology in oocytes was used to assess persistent functional changes after exposing cysteine-substituted receptors to methyl-MTS through n-decyl-MTS. Overlap of modified cysteine sidechains with bound anesthetic was inferred when anesthetic co-application with alkyl-MTS reagent blocked the development of persistent effects. In 1L232C32L receptors, only pentyl-MTS and hexyl-MTS induced persistent effects that were unaltered by etomidate co-application, precluding a direct estimate of intermolecular distance. In 1L232W3M286C2L receptors, sidechain overlap with bound etomidate was inferred for modifications with ethyl-MTS through n-pentyl-MTS, with unambiguous cut-on and cut-off. Comparison with results in 13M286C2L reveals that 1L232W, which increases maximal sidechain length by 2.1 Å, displaces etomidate closer to 3M286C by about 1.3 Å. Propofol results largely mirrored those with etomidate. These findings indicate that both etomidate and propofol bind within 1 Å of 1L232, consistent with cryo-EM structures. SIGNIFICANCE STATEMENT: We combined electrophysiology, cysteine substitutions, and n-alkyl-methanethiosulfonate modifiers in functional GABA receptors to enable precise estimates of the distance between β3M286C sidechains and anesthetics (etomidate and propofol) bound in transmembrane +/ inter-subunit pockets. Comparing results in α1β3M286Cγ2L and α1L232Wβ3M286Cγ2L receptors reveals that α1L232W mutations displace both anesthetics toward β3M286C, indicating that these anesthetics bind within 1 Å of the α1L232 sidechain in functional receptors, consistent with cryogenic electron microscopy structures derived under nonphysiologic conditions.
多种方法，包括低温电子显微镜（cryo-EM），表明麻醉剂依托咪酯和丙泊酚通过结合在 M286 和 L232 侧链附近的重叠跨膜亚基间位点来调节 12/32 GABA 受体。需要使用高精度方法在功能性受体中进行比较与 cryo-EM。我们之前使用取代的半胱氨酸修饰和保护（SCAMP）与 n-烷基-甲硫磺酸酯（MTS）试剂和 13M286C2L 受体中的电生理学来估计依托咪酯与 3M286 之间的距离，精度接近 1.3 Å。在这里，我们使用这种方法解决了三个更具体的目标：（i）在 1L232C32L 受体中测试了依托咪酯的 SCAMP；（ii）在 1L232W3M286C2L 受体中的研究评估了 1L232W 是否相对于 3M286C 取代了依托咪酯；（iii）将丙泊酚的结果与依托咪酯的结果进行了比较。卵母细胞中的电压钳电生理学用于评估将半胱氨酸取代的受体暴露于通过 n-癸基-MTS 的甲基-MTS 后持续功能变化。当烷基-MTS 试剂与麻醉剂共同应用时阻断持续效应的发展时，推断修饰的半胱氨酸侧链与结合麻醉剂重叠。在 1L232C32L 受体中，只有戊基-MTS 和己基-MTS 诱导的持续效应不受依托咪酯共同应用的影响，从而排除了直接估计分子间距离的可能性。在 1L232W3M286C2L 受体中，推断出乙基-MTS 至 n-戊基-MTS 的修饰与结合的依托咪酯之间存在侧链重叠，具有明确的截止和截止。与 13M286C2L 的结果进行比较表明，增加最大侧链长度 2.1 Å 的 1L232W 将依托咪酯更靠近 3M286C 取代约 1.3 Å。丙泊酚的结果在很大程度上反映了依托咪酯的结果。这些发现表明，依托咪酯和丙泊酚都结合在β3M286C 侧链的 1 Å 内，与 cryo-EM 结构一致。意义陈述：我们结合了电生理学、半胱氨酸取代和功能性 GABA 受体中的 n-烷基-甲硫磺酸酯修饰剂，以能够精确估计β3M286C 侧链与结合在跨膜+/亚基间口袋中的麻醉剂（依托咪酯和丙泊酚）之间的距离。比较在 α1β3M286Cγ2L 和 α1L232Wβ3M286Cγ2L 受体中的结果表明，α1L232W 突变使两种麻醉剂都朝向β3M286C 位移，这表明这些麻醉剂在功能性受体中结合在α1L232 侧链的 1 Å 内，与低温电子显微镜结构一致根据非生理条件得出的结构。
