Castel H, Louiset E, Anouar Y, Le Foll F, Cazin L, Vaudry H
European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Médicale (INSERM U 413), University of Rouen, Mont-Saint-Aignan, France.
J Neuroendocrinol. 2000 Jan;12(1):41-52. doi: 10.1046/j.1365-2826.2000.00417.x.
The effects of protein tyrosine kinase (PTK) and PTK inhibitors on the GABAA receptor function were studied in cultured frog pituitary melanotrophs by using the patch-clamp technique. Extracellular application of the PTK inhibitors genistein (10-9 to 10-5 M) or lavendustin A (10-12 to 10-7 M) provoked a bell-shaped potentiation of the whole-cell current induced by GABA (3x10-6 M). In contrast, at high concentrations, genistein (10-4 M) and lavendustin A (10-5 M) reversibly reduced the GABA-evoked current. Daidzein and lavendustin B, the inactive analogs of genistein and lavendustin A, respectively, did not modify the current induced by GABA. In the inside-out configuration, bath application of the recombinant PTK pp60c-src (75 U/ml) inhibited the GABA-activated chloride current, and the inhibitory effect of pp60c-src was prevented by genistein (10-7 M). Immunoblotting revealed that genistein, at doses of 10-7 M or 10-4 M, markedly inhibited tyrosine phosphorylation of the beta2/beta3 subunits of the GABAA receptor. Extracellular application of the PKA activator Bt2cAMP (10-3 M), the PKA/PKC inhibitor H7 (10-5 M) and the Cam KII inhibitor W7 (10-5 M) reversibly diminished the whole-cell GABA-induced current. Internal application of H7 and W7 (10-4 M) did not modify the dose-dependent effects of genistein. Internal application of sodium orthovanadate (10-4 M), a protein tyrosine phosphatase inhibitor, decreased the GABA-evoked current and markedly reduced the potentiating effect of genistein. The present study provides the first evidence that, in frog pituitary melanotrophs, the GABAA receptor is phosphorylated at least on its beta2/beta3 subunits by an endogenous PTK. Our data also demonstrate that tyrosine phosphorylation exerts an inhibitory effect on GABAA receptor function.
采用膜片钳技术，在培养的青蛙垂体黑素细胞中研究了蛋白酪氨酸激酶（PTK）及其抑制剂对GABAA受体功能的影响。细胞外施加PTK抑制剂染料木黄酮（10-9至10-5 M）或拉文杜斯汀A（10-12至10-7 M），可引起由γ-氨基丁酸（3×10-6 M）诱导的全细胞电流呈钟形增强。相反，在高浓度下，染料木黄酮（10-4 M）和拉文杜斯汀A（10-5 M）可使γ-氨基丁酸诱发的电流可逆性降低。染料木黄酮和拉文杜斯汀A的无活性类似物大豆苷元和拉文杜斯汀B，均未改变γ-氨基丁酸诱导的电流。在膜内面向外模式下，浴槽中加入重组PTK pp60c-src（75 U/ml）可抑制γ-氨基丁酸激活的氯离子电流，染料木黄酮（10-7 M）可阻止pp60c-src的抑制作用。免疫印迹显示，剂量为10-7 M或10-4 M的染料木黄酮可显著抑制GABAA受体β2/β3亚基的酪氨酸磷酸化。细胞外施加蛋白激酶A（PKA）激活剂Bt2cAMP（10-3 M）、PKA/蛋白激酶C（PKC）抑制剂H7（10-5 M）和钙调蛋白激酶II（Cam KII）抑制剂W7（10-5 M），可使全细胞γ-氨基丁酸诱导的电流可逆性减弱。细胞内施加H7和W7（10-4 M）不改变染料木黄酮的剂量依赖性效应。细胞内施加蛋白酪氨酸磷酸酶抑制剂原钒酸钠（10-4 M）可降低γ-氨基丁酸诱发的电流，并显著降低染料木黄酮的增强作用。本研究首次证明，在青蛙垂体黑素细胞中，GABAA受体至少在其β2/β3亚基上被内源性PTK磷酸化。我们的数据还表明，酪氨酸磷酸化对GABAA受体功能发挥抑制作用。
