Maintenance of recombinant type A gamma-aminobutyric acid receptor function: role of protein tyrosine phosphorylation and calcineurin.
Maintenance of recombinant type A gamma-aminobutyric acid receptor function: role of protein tyrosine phosphorylation and calcineurin.
复制标题
DOI：
--
复制
发表时间：
1998-07
期刊：
影响因子：
--
通讯作者：
Renqi Huang;G. Dillon
中科院分区：
文献类型：
--
作者：
Renqi Huang;G. Dillon
在本研究中，rundown的γ-氨基丁酸（GABA）激活的氯离子通道研究在重组GABAA受体稳定表达在人胚肾细胞（HEK 293），与传统的全细胞和白藜芦醇B穿孔补丁记录。当[ATP]i降低到1 mM，静息[Ca++]i缓冲到相对较高的水平时，α 3 β 2 γ 2 GABAA受体对相对较低[GABA]（高达50 μ M）的反应在全细胞构型中未显示出下降。然而，高[GABA]（大于200 μ M）诱导显着的下降，这是观察到的最大GABA诱导电流和GABA EC 50的下降。用含有4 mM Mg（++）-ATP和低静息[Ca++]i的溶液或在穿孔斑片记录期间完全防止Rundown。α 1 β 2 γ 2和β 2 γ 2受体的下降幅度相当。无论是刺激还是抑制蛋白激酶A或蛋白激酶C有一个显着的效果。然而，偏钒酸钠，蛋白酪氨酸磷酸酶的抑制剂，显着减少了流失。此外，抑制蛋白酪氨酸激酶活性的染料木黄酮或lavelustin A诱导的GABA反应的下降。抑制Ca++/钙调素依赖性磷酸酶钙调磷酸酶与氰戊菊酯也防止了对GABA的反应的下降。我们的研究结果表明，GABAA受体功能的下降是浓度依赖性的，由于ATP和/或未缓冲的[Ca++]i的消耗，并不依赖于α亚基的存在或亚型。我们建议，蛋白磷酸化在酪氨酸激酶依赖的网站，和一个独特的身份不明的网站，这是去磷酸化的钙调磷酸酶，保持GABAA受体的功能。
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.