Huang R Q, Dillon G H
Department of Pharmacology, University of North Texas Health Science Center at Fort Worth, USA.
J Pharmacol Exp Ther. 1998 Jul;286(1):243-55.
In the present study, rundown of gamma-aminobutyric acid (GABA)-activated Cl- channels was studied in recombinant GABAA receptors stably expressed in human embryonic kidney cells (HEK 293), with conventional whole-cell and amphotericin B-perforated patch recording. When [ATP]i was lowered to 1 mM and resting [Ca++]i was buffered to a relatively high level, the response of alpha 3 beta 2 gamma 2 GABAA receptors to relatively low [GABA] (up to 50 microM) did not show rundown in the whole-cell configuration. However, high [GABA] (greater than 200 microM) induced significant rundown, which was observed by decreases in both the maximum GABA-induced current and GABA EC50. Rundown was prevented completely with a solution containing 4 mM Mg(++)-ATP and low resting [Ca++]i, or during perforated patch recording. The magnitude of rundown was comparable in alpha 1 beta 2 gamma 2 and beta 2 gamma 2 receptors. Neither stimulation nor inhibition of protein kinase A or protein kinase C had a significant effect on rundown. However, sodium metavanadate, an inhibitor of protein tyrosine phosphatase, significantly reduced rundown. In addition, inhibition of protein tyrosine kinase activity by either genistein or lavendustin A induced rundown of the GABA response. Inhibition of the Ca++/calmodulin-dependent phosphatase calcineurin with fenvalerate also prevented rundown of the response to GABA. Our results demonstrate that rundown of GABAA receptor function is concentration-dependent, due to depletion of ATP and/or unbuffered [Ca++]i, and does not depend on the presence or subtype of the alpha subunit. We propose that protein phosphorylation at a tyrosine kinase-dependent site, and a distinct unidentified site, which is dephosphorylated by calcineurin, maintains the function of GABAA receptors.
在本研究中，采用传统的全细胞膜片钳和两性霉素B穿孔膜片钳记录技术，对稳定表达于人类胚胎肾细胞（HEK 293）中的重组GABAA受体上γ-氨基丁酸（GABA）激活的氯离子通道的衰减情况进行了研究。当细胞内ATP浓度降至1 mM且静息状态下的细胞内钙离子浓度被缓冲至相对较高水平时，α3β2γ2 GABAA受体对相对低浓度GABA（高达50 μM）的反应在全细胞模式下未表现出衰减。然而，高浓度GABA（大于200 μM）会诱导显著的衰减，这可通过最大GABA诱导电流和GABA半数有效浓度（EC50）的降低来观察到。含有4 mM Mg2+-ATP且静息细胞内钙离子浓度较低的溶液，或在穿孔膜片钳记录过程中，可完全防止衰减。α1β2γ2和β2γ2受体的衰减程度相当。蛋白激酶A或蛋白激酶C的刺激或抑制对衰减均无显著影响。然而，蛋白酪氨酸磷酸酶抑制剂偏钒酸钠可显著减少衰减。此外，染料木黄酮或拉文杜斯汀A对蛋白酪氨酸激酶活性的抑制会诱导GABA反应的衰减。用氰戊菊酯抑制钙离子/钙调蛋白依赖性磷酸酶钙调神经磷酸酶也可防止对GABA反应的衰减。我们的结果表明，GABAA受体功能的衰减是浓度依赖性的，这是由于ATP的消耗和/或未缓冲的细胞内钙离子浓度所致，且不依赖于α亚基的存在或亚型。我们提出，酪氨酸激酶依赖性位点以及一个由钙调神经磷酸酶去磷酸化的独特未确定位点的蛋白磷酸化维持了GABAA受体的功能。
