Glutamate transporter GLAST/EAAT1 directs cell surface expression of FXYD2/γ subunit of Na, K-ATPase in human fetal astrocytes
Glutamate transporter GLAST/EAAT1 directs cell surface expression of FXYD2/γ subunit of Na, K-ATPase in human fetal astrocytes
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DOI：
10.1016/j.neuint.2006.12.015
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发表时间：
2007-06-01
影响因子：
4.2
通讯作者：
Gegelashvili, Georgi
中科院分区：
文献类型：
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作者：
Gegelashvili, Marina;Rodriguez-Kern, Anna;Gegelashvili, Georgi
星形胶质细胞中兴奋性神经递质谷氨酸的Na+依赖性摄取增加细胞能量需求，主要是由于谷氨酰胺合成酶和Na+，K+-ATP酶的ATP消耗增加。GLAST/EAAT 1是未分化培养物中人胎儿星形胶质细胞表达的唯一谷氨酸转运子亚型，其主要库仅限于细胞质区室。升高的谷氨酸浓度（高达50 μ M）刺激谷氨酸摄取和Na+，K+-ATP酶活性，并伴随着增加细胞表面的GLAST和FXYD 2/γ亚基的Na+，K+-ATP酶的表达。谷氨酸或其代谢产物本身的细胞内积累是不负责这些变化，因为代谢惰性的运输底物，D-天冬氨酸，发挥同样的作用。纳摩尔浓度的TFB-TBOA，一种新型的非转运谷氨酸载体抑制剂，几乎完全逆转了谷氨酸或D-天冬氨酸的作用。在同样的条件下（即阻断谷氨酸转运），一种强有力的Na+离子载体莫能菌素对Na+，K+-ATP酶的激活和细胞表面γ亚基或GLAST的表达均无显著影响。为了阐明γ亚基在谷氨酸摄取依赖性运输事件或星形胶质细胞钠泵激活中的作用，在一些培养物中，使用siRNA沉默有效地敲低γ亚基/FXYD 2。与TFB-TBOA的阻断作用不同，γ亚基的下调对GLAST的运输和活性都没有影响。然而，γ亚基的缺失有效地消除了Na+，K+-ATP酶的谷氨酸摄取依赖性激活。从培养物中撤回siRNA后，γ亚基的表达水平和Na+，K+-ATP酶对谷氨酸/天冬氨酸摄取的敏感性已同时恢复。因此，GLAST的活性将FXYD 2蛋白/-γ亚基引导至细胞表面，这进而导致星形胶质细胞钠泵的激活，推测是由于γ亚基对Na+，K+-ATP酶的催化α亚基的动力学参数的调节作用。(C)2007爱思唯尔有限公司保留所有权利。
Na+-dependent uptake of excitatory neurotransmitter glutamate in astrocytes increases cell energy demands primarily due to the elevated ATP consumption by glutamine synthetase and Na+, K+-ATPase. The major pool of GLAST/EAAT1, the only glutamate transporter subtype expressed by human fetal astrocytes in undifferentiated cultures, was restricted to the cytoplasmic compartment. Elevated glutamate concentrations (up to 50 mu M) stimulated both glutamate uptake and Na+, K+-ATPase activity and concomitantly increased cell surface expression of GLAST and FXYD2/gamma subunit of Na+, K+-ATPase. Intracellular accumulation of glutamate or its metabolites per se was not responsible for these changes since metabolically inert transport substrate, D-aspartate, exerted the same effect. Nanomolar concentrations of TFB-TBOA, a novel nontransportable inhibitor of glutamate carriers, almost completely reversed the action of glutamate or D-aspartate. In the same conditions (i.e. block of glutamate transport) monensin, a potent Na+ ionophore, had no significant effect neither on the activation of Na+, K+-ATPase nor on the cell surface expression of gamma subunit or GLAST. In order to elucidate the roles of gamma subunit in the glutamate uptake-dependent trafficking events or the activation of the astroglial sodium pump, in some cultures gamma subunit/FXYD2 was effectively knocked down using siRNA silencing. Unlike the blocking effect of TFB-TBOA, the down-regulation of gamma subunit had no effect neither on the trafficking nor activity of GLAST. However, the loss of gamma subunit effectively abolished the glutamate uptake-dependent activation of Na+, K+-ATPase, Following withdrawal of siRNA from cultures, the expression levels of gamma subunit and the sensitivity of Na+, K+-ATPase to glutamate/aspartate uptake have been concurrently restored. Thus, the activity of GLAST directs FXYD2 protein/-gamma subunit to the cell surface, that, in turn, leads to the activation of the astroglial sodium pump, presumably due to the modulatory effect of gamma subunit on the kinetic parameters of catalytic a subunit(s) of Na+, K+-ATPase. (C) 2007 Elsevier Ltd. All rights reserved.