Department of Physiology, University of Tübingen, Tübingen, Germany.
J Neurochem. 2010 Jun;113(6):1426-35. doi: 10.1111/j.1471-4159.2010.06678.x. Epub 2010 Mar 10.
The glutamate transporters EAAT3 and EAAT4 are expressed in neurons. They contribute to the cellular uptake of glutamate and aspartate and thus to the clearance of the excitatory transmitters from the extracellular space. During ischemia, extracellular accumulation of glutamate may trigger excitotoxicity. Energy depletion leads to activation of the AMP-activated protein kinase (AMPK), a kinase enhancing energy production and limiting energy expenditure. The present study thus explored the possibility that AMPK regulates EAAT3 and/or EAAT4. To this end, EAAT3 or EAAT4 were expressed in Xenopus oocytes with or without AMPK and electrogenic glutamate transport determined by dual electrode voltage clamp. In EAAT3- and in EAAT4-expressing oocytes glutamate generated a current (I(g)), which was half maximal (K(M)) at 74 microM (EAAT3) or at 4 microM (EAAT4) glutamate. Co-expression of constitutively active (gammaR70Q)AMPK or of wild type AMPK did not affect K(M) but significantly decreased the maximal I(g) in both EAAT3- (by 34%) and EAAT4- (by 49%) expressing oocytes. Co-expression of the inactive mutant (alphaK45R)AMPK [alpha1(K45R)beta1gamma1] did not appreciably affect I(g). According to confocal microscopy and chemiluminescence co-expression of (gammaR70Q)AMPK or of wild type AMPK reduced the membrane abundance of EAAT3 and EAAT4. The observations show that AMPK down-regulates Na(+)-coupled glutamate transport.
谷氨酸转运体 EAAT3 和 EAAT4 表达于神经元中。它们有助于细胞摄取谷氨酸和天冬氨酸，从而清除细胞外间隙中的兴奋性递质。在缺血期间，细胞外谷氨酸的积累可能引发兴奋性毒性。能量耗竭导致 AMP 激活的蛋白激酶（AMPK）的激活，该激酶增强能量产生并限制能量消耗。因此，本研究探讨了 AMPK 是否调节 EAAT3 和/或 EAAT4 的可能性。为此，用 AMPK 或不用 AMPK 在非洲爪蟾卵母细胞中表达 EAAT3 或 EAAT4，并通过双电极电压钳测定电活性谷氨酸转运。在 EAAT3 和 EAAT4 表达的卵母细胞中，谷氨酸产生电流（I(g)），谷氨酸的半最大值（K(M)）为 74 microM（EAAT3）或 4 microM（EAAT4）。组成激活的（gammaR70Q）AMPK 或野生型 AMPK 的共表达不影响 K(M)，但显著降低了 EAAT3-（34%）和 EAAT4-（49%）表达卵母细胞的最大 I(g)。不活跃的突变体（alphaK45R）AMPK [alpha1(K45R)beta1gamma1] 的共表达对 I(g)没有明显影响。根据共聚焦显微镜和化学发光，（gammaR70Q）AMPK 或野生型 AMPK 的共表达减少了 EAAT3 和 EAAT4 的膜丰度。这些观察结果表明，AMPK 下调了 Na(+)-偶联的谷氨酸转运。
