4'-氯地西泮和β-咔啉-3-羧酸酯引发的γ-氨基丁酸受体负变构调节差异：天然受体与重组受体的研究
Differences in the negative allosteric modulation of gamma-aminobutyric acid receptors elicited by 4'-chlorodiazepam and by a beta-carboline-3-carboxylate ester: a study with natural and reconstituted receptors.
作者信息
Puia G, Santi M R, Vicini S, Pritchett D B, Seeburg P H, Costa E
机构信息
FIDIA-Georgetown Institute for the Neurosciences, Georgetown University, Washington, DC 20007.
出版信息
Proc Natl Acad Sci U S A. 1989 Sep;86(18):7275-9. doi: 10.1073/pnas.86.18.7275.
Cl- currents elicited by gamma-aminobutyric acid (GABA) application were recorded with the whole-cell tight-seal technique from voltage-clamped cortical neurons of neonatal rats in primary culture. The peripheral benzodiazepine recognition site ligand 4'-chlorodiazepam [Ro 5-4864; 7-chloro-1,3-dihydro-1-methyl-5-(4-chlorophenyl)-2H-[1,4]-benzodiazep in-2- one] inhibited the GABA-generated currents in a dose-dependent manner. Also, a beta-carboline (DMCM; 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate methyl ester), acting as a negative allosteric modulator of GABAA receptors, reduced the intensity of GABA-generated currents with similar efficacy but greater potency. Flumazenil (Ro 15-1788; 8-fluro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-[1,5-a] [1,4]-benzodiazepine-3-carboxylate ethyl ester) antagonized DMCM inhibition but not that elicited by 4'-chlorodiazepam. The isoquinoline carboxamide PK 11195, an antagonist of 4'-chlorodiazepam effects in other systems, failed to antagonize the action of 4'-chlorodiazepam. The transient expression of various molecular forms of GABAA receptors in the human embryonic kidney cell line 293 allowed a study of the minimal structural requirements for the inhibition of GABA-induced Cl- currents by bicuculline, picrotoxin, 4'-chlorodiazepam, and DMCM. GABA-elicited Cl- currents in cells coexpressing alpha 1 and beta 1 subunits of GABAA receptors were inhibited by bicuculline and picrotoxin, but not by DMCM or 4'-chlorodiazepam. Conversely, the GABA currents in cells coexpressing alpha 1 beta 1 and gamma 2 subunits were inhibited by bicuculline, picrotoxin, 4'-chlorodiazepam, and DMCM. Since the Cl- currents generated by GABA in some molecular forms of GABAA receptors are inhibited by bicuculline and picrotoxin only, 4'-chlorodiazepam cannot be acting isosterically with picrotoxin.
采用全细胞膜片钳技术，在电压钳制下，从原代培养的新生大鼠皮质神经元中记录γ-氨基丁酸（GABA）诱发的Cl⁻电流。外周苯二氮䓬识别位点配体4'-氯地西泮[Ro 5-4864；7-氯-1,3-二氢-1-甲基-5-(4-氯苯基)-2H-[1,4]-苯并二氮杂䓬-2-酮]以剂量依赖方式抑制GABA产生的电流。此外，作为GABAA受体负性变构调节剂的β-咔啉（DMCM；6,7-二甲氧基-4-乙基-β-咔啉-3-羧酸甲酯），以相似的效能但更高的效价降低GABA产生电流的强度。氟马西尼（Ro 15-1788；8-氟-5,6-二氢-5-甲基-6-氧代-4H-咪唑并[1,5-a][1,4]-苯并二氮杂䓬-3-羧酸乙酯）拮抗DMCM的抑制作用，但不拮抗4'-氯地西泮引起的抑制作用。异喹啉甲酰胺PK 11195，在其他系统中是4'-氯地西泮作用的拮抗剂，未能拮抗4'-氯地西泮 的作用。在人胚肾细胞系293中瞬时表达各种分子形式的GABAA受体，使得能够研究荷包牡丹碱、印防己毒素、4'-氯地西泮和DMCM抑制GABA诱导的Cl⁻电流的最小结构要求。在共表达GABAA受体α1和β1亚基的细胞中，GABA诱发的Cl⁻电流被荷包牡丹碱和印防己毒素抑制，但不被DMCM或4'-氯地西泮抑制。相反，在共表达α1β1和γ2亚基的细胞中，GABA电流被荷包牡丹碱、印防己毒素、4'-氯地西泮和DMCM抑制。由于在某些分子形式的GABAA受体中，GABA产生的Cl⁻电流仅被荷包牡丹碱和印防己毒素抑制，所以4'-氯地西泮不能与印防己毒素起等排作用。
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