Robello M, Amico C, Cupello A
INFM Unità di Genova, Dipartimento di Fisica, Università di Genova, Italy.
Eur Biophys J. 1997;25(3):181-7. doi: 10.1007/s002490050030.
The function of the GABAA receptor has been studied using the whole cell voltage clamp recording technique in rat cerebellum granule cells in culture. Activation of NMDA-type glutamate receptors causes a reduction in the effect of GABA. Full GABAA receptor activity was recovered after washing out NMDA and NMDA action was prevented in a Mg+2 containing medium. The NMDA effect was also absent when extracellular Ca+2 was replaced by Ba+2 and when 10 mM Bapta was present in the intracellular solution. Charge accumulations via voltage activated Ca+2 channels greater than the ones via NMDA receptors do not cause any reduction in GABAA receptor function, suggesting that Ca+2 influx through NMDA receptor channels is critical for the effect. The NMDA effect was reduced by including adenosine-5'-O-3-thiophosphate (ATP-gamma-S) in the internal solution and there was a reduction in the NMDA effect caused by deltamethrin, a calcineurin inhibitor. Part of the NMDA induced GABAA receptor impairment was prevented by prior treatment with L-arginine. Analogously, part of the NMDA effect was prevented by blockage of NO-synthase activity by N omega-nitro-L-arginine. A combination of NO-synthase and calcineurin inhibitors completely eliminated the NMDA action. An analogous result was obtained by combining the NO-synthase inhibitor with the addition of ATP-gamma-S to the pipette medium. The additivity of the prevention of the NMDA impairment of GABAA receptor by blocking the L-arginine/NO pathway and inhibiting calcineurin activity suggests an independent involvement of these two pathways in the interaction between NMDA and the GABAA receptor. On the one hand Ca+2 influx across NMDA channels activates calcineurin and dephosphorylates the GABAA receptor complex directly or dephosphorylates proteins critical for the function of the receptor. On the other hand, Ca+2 influx activates NO-synthase and induces nitric oxide production, which regulates such receptors via protein kinase G activity.
已使用全细胞电压钳记录技术在培养的大鼠小脑颗粒细胞中研究了GABAA受体的功能。NMDA型谷氨酸受体的激活导致GABA作用减弱。洗去NMDA后，GABAA受体的全部活性得以恢复，并且在含Mg+2的培养基中可防止NMDA的作用。当细胞外Ca+2被Ba+2取代以及细胞内溶液中存在10 mM Bapta时，NMDA的作用也不存在。通过电压激活的Ca+2通道的电荷积累大于通过NMDA受体的电荷积累，不会导致GABAA受体功能的任何降低，这表明通过NMDA受体通道的Ca+2内流对该作用至关重要。通过在内部溶液中加入腺苷-5'-O-3-硫代磷酸酯（ATP-γ-S），NMDA的作用减弱，并且由钙调神经磷酸酶抑制剂溴氰菊酯引起的NMDA作用也减弱。预先用L-精氨酸处理可部分预防NMDA诱导的GABAA受体损伤。类似地，通过Nω-硝基-L-精氨酸阻断一氧化氮合酶活性可部分预防NMDA的作用。一氧化氮合酶和钙调神经磷酸酶抑制剂的组合完全消除了NMDA的作用。通过将一氧化氮合酶抑制剂与向移液管培养基中添加ATP-γ-S相结合，获得了类似的结果。通过阻断L-精氨酸/一氧化氮途径和抑制钙调神经磷酸酶活性来预防NMDA对GABAA受体的损伤的相加性表明，这两条途径在NMDA与GABAA受体之间的相互作用中独立起作用。一方面，通过NMDA通道的Ca+2内流激活钙调神经磷酸酶，并直接使GABAA受体复合物去磷酸化或使对受体功能至关重要的蛋白质去磷酸化。另一方面，Ca+2内流激活一氧化氮合酶并诱导一氧化氮生成，一氧化氮通过蛋白激酶G活性调节此类受体。
