Gyenes M, Wang Q, Gibbs T T, Farb D H
Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Massachusetts 02118.
Mol Pharmacol. 1994 Sep;46(3):542-9.
Whole-cell and patch-voltage clamp experiments were carried out on cultured chick spinal cord neurons to investigate the dependence of gamma-aminobutyric acid (GABA)A receptor function on intracellular phosphorylation factors. Without ATP in the intracellular solution, repeated application of 30 microM GABA results in a progressive decline (run-down) of the currents evoked by GABA in standard whole-cell recordings but not when the nystatin-perforated patch method is used. Run-down is also observed in outside-out excised patch recordings, indicating that any enzymatic factors required for run-down must be closely associated with the plasma membrane. Run-down is associated with decreases in both the maximum GABA-induced current and the GABA EC50. Inclusion of magnesium adenosine-5'-O-(3-thio)triphosphate in the intracellular buffer prevents the decline in the maximum GABA response but the GABA EC50 still decreases, resulting in a "run-up" of the response at low (3 microM) GABA concentrations. Run-down is use dependent, requiring repeated activation of the GABAA receptor by high (30 microM) GABA concentrations. However, use-independent run-down can be induced by the inclusion of alkaline phosphatase in the intracellular buffer. The response to 3 microM GABA does not normally run down, but run-down is observed when the response to 3 microM GABA is potentiated with pentobarbital or allopregnanolone, suggesting that run-down is consequence of GABA receptor activation and/or desensitization. Run-down of the potentiated GABA response can be prevented by addition of magnesium adenosine-5'-O-(3-thio)triphosphate to the intracellular solution. Strikingly, run-down results in a significant decrease in the potentiating effects of positive modulators, whereas the inhibitory effects of negative modulators such as pregnenolone sulfate and ZnCl2 are unchanged. The results demonstrate that phosphorylation factors have the capacity to control GABAA receptor pharmacology, affecting the potency and efficacy of GABA, the kinetics of GABAA receptor desensitization, and the sensitivity of the receptor to modulators such as steroids, benzodiazepines, and barbiturates.
在培养的鸡脊髓神经元上进行了全细胞和膜片钳电压钳实验，以研究γ-氨基丁酸（GABA）A受体功能对细胞内磷酸化因子的依赖性。细胞内溶液中无ATP时，在标准全细胞记录中重复施加30μM GABA会导致GABA诱发的电流逐渐下降（衰减），但使用制霉菌素穿孔膜片法时则不会。在外侧向外膜片钳记录中也观察到衰减，这表明衰减所需的任何酶因子必须与质膜紧密相关。衰减与最大GABA诱导电流和GABA EC50的降低有关。细胞内缓冲液中加入镁腺苷-5'-O-（3-硫代）三磷酸可防止最大GABA反应的下降，但GABA EC50仍会降低，导致在低（3μM）GABA浓度下反应出现“增强”。衰减是使用依赖性的，需要高（30μM）GABA浓度反复激活GABAA受体。然而，细胞内缓冲液中加入碱性磷酸酶可诱导非使用依赖性衰减。对3μM GABA的反应通常不会衰减，但当用戊巴比妥或别孕烯醇酮增强对3μM GABA的反应时会观察到衰减，这表明衰减是GABA受体激活和/或脱敏的结果。向细胞内溶液中加入镁腺苷-5'-O-（3-硫代）三磷酸可防止增强的GABA反应衰减。令人惊讶的是，衰减导致正性调节剂的增强作用显著降低，而硫酸孕烯醇酮和ZnCl2等负性调节剂的抑制作用不变。结果表明，磷酸化因子有能力控制GABAA受体药理学，影响GABA的效力和效能、GABAA受体脱敏动力学以及受体对类固醇、苯二氮䓬类和巴比妥类等调节剂的敏感性。
