Fantasia Ryan J, Nourmahnad Anahita, Halpin Elizabeth, Forman Stuart A
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. 2021 Jun;99(6):426-434. doi: 10.1124/molpharm.120.000224. Epub 2021 Mar 25.
The anesthetic etomidate modulates synaptic 12/32 GABA receptors via binding sites located in transmembrane +/- interfaces. Various approaches indicate that etomidate binds near 2/3M286 side chains, including recent cryogenic electron microscopy images in 122L receptors under nonphysiologic conditions with ∼3.5-Å resolution. We hypothesized that substituted cysteine modification and protection experiments using variably sized -alkyl-methanethiosulfonate (MTS) reagents could precisely estimate the distance between bound etomidate and 3M286 side chains in activated functional receptors. Using voltage-clamp electrophysiology in oocytes expressing 13M286C2L GABA receptors, we measured functional changes after exposing GABA-activated receptors to -alkyl-MTS reagents, from methyl-MTS to -decyl-MTS. Based on previous studies using a large sulfhydryl reagent, we anticipated that cysteine modifications large enough to overlap etomidate sites would cause persistently increased GABA sensitivity and decreased etomidate modulation and that etomidate would hinder these modifications, reducing effects. Based on altered GABA or etomidate sensitivity, ethyl-MTS and larger -alkyl-MTS reagents modified GABA-activated 13M286C2L GABA receptors. Receptor modification by -propyl-MTS or larger reagents caused persistently increased GABA sensitivity and decreased etomidate modulation. Receptor-bound etomidate blocked 3M286C modification by -propyl-MTS, -butyl-MTS, and -hexyl-MTS. In contrast, GABA sensitivity was unaltered by receptor exposure to methyl-MTS or ethyl-MTS, and ethyl-MTS modification uniquely increased etomidate modulation. These results reveal a "cut-on" between ethyl-MTS and -propyl-MTS, from which we infer that --(-propyl) is the smallest 3M286C appendage that overlaps with etomidate sites. Molecular models of the native methionine and --ethyl and --(-propyl) modified cysteines suggest that etomidate is located between 1.7 and 3.0 Å from the 3M286 side chain. SIGNIFICANCE STATEMENT: Precise spatial relationships between drugs and their receptor sites are essential for mechanistic understanding and drug development. This study combined electrophysiology, a cysteine substitution, and -alkyl-methanethiosulfonate modifiers, creating a precise molecular ruler to estimate the distance between a α1β3γ2L GABA type A receptor residue and etomidate bound in the transmembrane +/- interface.
麻醉药依托咪酯通过位于跨膜+/-界面的结合位点调节突触12/32型GABA受体。各种方法表明依托咪酯结合在2/3M286侧链附近，包括最近在非生理条件下分辨率约为3.5埃的122L受体中的低温电子显微镜图像。我们假设使用大小可变的烷基甲硫基磺酸盐（MTS）试剂进行的半胱氨酸取代修饰和保护实验可以精确估计结合的依托咪酯与活化的功能性受体中3M286侧链之间的距离。在表达13M286C2L GABA受体的卵母细胞中使用电压钳电生理学，我们测量了将GABA激活的受体暴露于从甲基-MTS到癸基-MTS的烷基-MTS试剂后的功能变化。基于先前使用大巯基试剂的研究，我们预计足够大以与依托咪酯位点重叠的半胱氨酸修饰会导致GABA敏感性持续增加和依托咪酯调节作用降低，并且依托咪酯会阻碍这些修饰，从而降低其效果。基于GABA或依托咪酯敏感性的改变，乙基-MTS和更大的烷基-MTS试剂修饰了GABA激活的13M286C2L GABA受体。丙基-MTS或更大试剂对受体的修饰导致GABA敏感性持续增加和依托咪酯调节作用降低。受体结合的依托咪酯阻断了丙基-MTS、丁基-MTS和己基-MTS对3M286C的修饰。相比之下，受体暴露于甲基-MTS或乙基-MTS时GABA敏感性未改变，并且乙基-MTS修饰独特地增加了依托咪酯调节作用。这些结果揭示了乙基-MTS和丙基-MTS之间的“界限”，由此我们推断-（-丙基）是与依托咪酯位点重叠的最小的3M286C附加基团。天然甲硫氨酸以及-乙基和-（-丙基）修饰的半胱氨酸的分子模型表明，依托咪酯位于距3M286侧链1.7至3.0埃之间。意义声明：药物与其受体位点之间精确的空间关系对于机理理解和药物开发至关重要。本研究结合了电生理学、半胱氨酸取代和烷基甲硫基磺酸盐修饰剂，创建了一把精确的分子尺来估计α1β3γ2L A型GABA受体残基与跨膜+/-界面中结合的依托咪酯之间的距离。
