Chapell R, Bueno O F, Alvarez-Hernandez X, Robinson L C, Leidenheimer N J
Department of Pharmacology, Louisiana State University Medical Center, Shreveport, Louisiana 71130, USA.
J Biol Chem. 1998 Dec 4;273(49):32595-601. doi: 10.1074/jbc.273.49.32595.
The inhibition of gamma-aminobutyric acid (GABA)-gated chloride currents by the protein kinase C (PKC) activator 4beta-phorbol 12-myristate 13-acetate (PMA) was investigated using recombinant human GABAA receptors expressed in Xenopus oocytes. PMA (5 nM) reduced the GABA response in oocytes expressing the alpha1 beta2 gamma2L receptor construct, as measured by the two-electrode voltage-clamp method. GABA responses declined to approximately 25% of their pretreatment value within 45 min. GABA responses in oocytes expressing a receptor construct from which the known PKC phosphorylation sites were absent, alpha1 beta2(S410A), were comparably inhibited. Phorbol 12-monomyristate (PMM; 5 nM), which does not activate PKC, did not alter the GABA response in either construct, while the PKC inhibitor calphostin C (0.5 microM) prevented the PMA effect. To further investigate PMA inhibition of the GABA response, a GABAA receptor alpha1 subunit/green fluorescent protein (GFP) chimera (alpha1GFP) was used to visualize GABAA receptor distribution. Similar to the wild type constructs, PMA robustly decreased GABA responses in oocytes expressing alpha1GFP beta2 gamma2L and alpha1GFP beta2(S410A) receptor constructs. Following PMA treatment, GFP fluorescence in the oocyte plasma membrane was decreased to approximately 45% of the pretreatment values indicating GABAA receptor internalization. This effect of PMA was prevented by calphostin C and was not produced by PMM. Experiments with bd24, a monoclonal antibody which recognizes an extracellular epitope of the alpha1 subunit, were used to demonstrate that PMA, but not PMM, decreases alpha1 subunit immunoreactivity in the plasma membrane of intact oocytes expressing the alpha1 beta2 gamma2L construct, thus confirming the results obtained with the chimeric receptor. It is concluded that, in Xenopus oocytes, PMA induces an internalization of the GABAA receptor through PKC-mediated phosphorylation of an unidentified protein(s) and that this contributes to the decrease in electrophysiological responses to GABA following PKC activation.
利用非洲爪蟾卵母细胞中表达的重组人GABAA受体，研究了蛋白激酶C（PKC）激活剂4β-佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）对γ-氨基丁酸（GABA）门控氯电流的抑制作用。采用双电极电压钳法测量，PMA（5 nM）降低了表达α1β2γ2L受体构建体的卵母细胞中的GABA反应。在45分钟内，GABA反应下降至预处理值的约25%。表达缺乏已知PKC磷酸化位点的受体构建体α1β2（S410A）的卵母细胞中的GABA反应受到类似抑制。不激活PKC的佛波醇12-单肉豆蔻酸酯（PMM；5 nM）对两种构建体中的GABA反应均无影响，而PKC抑制剂钙泊三醇C（0.5 μM）可阻止PMA的作用。为进一步研究PMA对GABA反应的抑制作用，使用GABAA受体α1亚基/绿色荧光蛋白（GFP）嵌合体（α1GFP）来观察GABAA受体的分布。与野生型构建体相似，PMA显著降低了表达α1GFPβ2γ2L和α1GFPβ2（S410A）受体构建体的卵母细胞中的GABA反应。PMA处理后，卵母细胞质膜中的GFP荧光降至预处理值的约45%，表明GABAA受体内化。钙泊三醇C可阻止PMA的这种作用，而PMM则不会产生这种作用。使用识别α1亚基细胞外表位的单克隆抗体bd24进行的实验表明，PMA而非PMM可降低表达α1β2γ2L构建体的完整卵母细胞质膜中的α1亚基免疫反应性，从而证实了嵌合受体实验的结果。得出的结论是，在非洲爪蟾卵母细胞中，PMA通过PKC介导的未鉴定蛋白的磷酸化诱导GABAA受体内化，这导致PKC激活后对GABA的电生理反应降低。
