Wingrove P B, Thompson S A, Wafford K A, Whiting P J
Neuroscience Research Centre, Merck Sharp & Dohme Research Laboratories, Harlow, Essex CM20 2QR, United Kingdom.
Mol Pharmacol. 1997 Nov;52(5):874-81. doi: 10.1124/mol.52.5.874.
Pharmacological analyses of gamma-aminobutyric acidA (GABAA) receptor subtypes have suggested that both the alpha and gamma subunits, but not the beta subunit, contribute to the benzodiazepine binding site. We took advantage of the different pharmacological properties conferred by the inclusion of different gamma subunits in the receptor macromolecule to identify amino acids gamma2Phe77 and gamma2Met130 as key determinants of the benzodiazepine binding site. gamma2Phe77 was required for high affinity binding of the benzodiazepine site ligands flumazenil, CL218,872, and methyl-beta-carboline-3-carboxylate but not flunitrazepam. This amino acid was, however, required for allosteric modulation by flunitrazepam, as well as other benzodiazepine site ligands. In contrast, gamma2Met130 was required for high affinity binding of flunitrazepam, clonazepam, and triazolam but not flumazenil, CL218, 872, or methyl-beta-carboline-3-carboxylate and did not affect benzodiazepine efficacy. Introduction of the phenylalanine and methionine into the appropriate positions of gamma1 was not sufficient to confer high affinity for the benzodiazepine site ligand zolpidem. These data show that gamma2Phe77 and gamma2Met130 are necessary for high affinity binding of a number of benzodiazepine site ligands. Although most previous studies have focused on the contribution of the alpha subunit, we demonstrated a critical role for the gamma subunit at the benzodiazepine binding site, indicating that this modulatory site is located at the interface of these two subunits. Furthermore, gamma2Phe77 is homologous to alpha1Phe64, which has been previously shown to be a key determinant of the GABA binding site, suggesting a conservation of motifs between different ligand binding sites on the GABAA receptor.
γ-氨基丁酸A（GABAA）受体亚型的药理学分析表明，α和γ亚基而非β亚基参与苯二氮䓬结合位点的构成。我们利用受体大分子中不同γ亚基所赋予的不同药理学特性，确定γ2苯丙氨酸77位和γ2蛋氨酸130位氨基酸是苯二氮䓬结合位点的关键决定因素。γ2苯丙氨酸77位是苯二氮䓬位点配体氟马西尼、CL218,872和甲基-β-咔啉-3-羧酸酯高亲和力结合所必需的，但对氟硝西泮不是必需的。然而，该氨基酸是氟硝西泮以及其他苯二氮䓬位点配体变构调节所必需的。相比之下，γ2蛋氨酸130位是氟硝西泮、氯硝西泮和三唑仑高亲和力结合所必需的，但对氟马西尼、CL218,872或甲基-β-咔啉-3-羧酸酯不是必需的，且不影响苯二氮䓬的效力。将苯丙氨酸和蛋氨酸引入γ1的适当位置不足以赋予对苯二氮䓬位点配体唑吡坦的高亲和力。这些数据表明，γ2苯丙氨酸77位和γ2蛋氨酸130位是多种苯二氮䓬位点配体高亲和力结合所必需的。尽管此前大多数研究集中于α亚基的作用，但我们证明了γ亚基在苯二氮䓬结合位点的关键作用，表明该调节位点位于这两个亚基的界面处。此外，γ2苯丙氨酸77位与α1苯丙氨酸64位同源，此前已证明α1苯丙氨酸64位是GABA结合位点的关键决定因素，这表明GABAA受体上不同配体结合位点之间存在基序保守性。
