The Density of EAAC1 (EAAT3) Glutamate Transporters Expressed by Neurons in the Mammalian CNS
The Density of EAAC1 (EAAT3) Glutamate Transporters Expressed by Neurons in the Mammalian CNS
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DOI：
10.1523/jneurosci.5347-11.2012
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发表时间：
2012-04-25
影响因子：
5.3
通讯作者：
Danbolt, Niels C.
中科院分区：
文献类型：
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作者：
Holmseth, Silvia;Dehnes, Yvette;Danbolt, Niels C.
兴奋性氨基酸的细胞外水平通过谷氨酸转运蛋白的作用保持较低。谷氨酸/天冬氨酸转运蛋白（GLAST）和谷氨酸转运蛋白-1（GLT-1）是最丰富的亚型，对哺乳动物CNS的功能至关重要，但EAAC 1亚型在突触谷氨酸清除中的作用仍有争议，因为该转运蛋白在不同组织中的密度尚未确定。我们使用纯化的EAAC 1蛋白作为免疫印迹过程中的标准，以测量EAAC 1在不同的中枢神经系统区域的浓度。最高的EAAC 1水平被发现在年轻的成年大鼠海马。这里，EAAC 1的浓度近似于0.013 mg/g组织（近似于130分子μ m（-3）），比GLT-1的浓度低100倍。不像GLT-1的表达，增加与电路的形成，只有轻微的变化，EAAC 1的浓度，观察到从E18到成年。在海马切片中，光解MNI-D-天冬氨酸（4-甲氧基-7-硝基吲哚啉-D-天冬氨酸）未能引起CA 1锥体神经元中EAAC 1介导的转运体电流，并且在脊髓中未检测到D-天冬氨酸摄取。使用EAAC 1基因敲除小鼠作为阴性对照，以建立抗体特异性，我们表明，这些相对少量的EAAC 1蛋白广泛分布在所有海马神经元的胞体和树突。这些发现提出了新的问题，即如此少的转运蛋白如何影响兴奋性突触上NMDA受体的激活。
The extracellular levels of excitatory amino acids are kept low by the action of the glutamate transporters. Glutamate/aspartate transporter ( GLAST) and glutamate transporter-1 (GLT-1) are the most abundant subtypes and are essential for the functioning of the mammalian CNS, but the contribution of the EAAC1 subtype in the clearance of synaptic glutamate has remained controversial, because the density of this transporter in different tissues has not been determined. We used purified EAAC1 protein as a standard during immunoblotting to measure the concentration of EAAC1 in different CNS regions. The highest EAAC1 levels were found in the young adult rat hippocampus. Here, the concentration of EAAC1 was similar to 0.013 mg/g tissue (similar to 130 molecules mu m(-3)), 100 times lower than that of GLT-1. Unlike GLT-1 expression, which increases in parallel with circuit formation, only minor changes in the concentration of EAAC1 were observed from E18 to adulthood. In hippocampal slices, photolysis of MNI-D-aspartate (4-methoxy-7-nitroindolinyl-D-aspartate) failed to elicit EAAC1-mediated transporter currents in CA1 pyramidal neurons, and D-aspartate uptake was not detected electron microscopically in spines. Using EAAC1 knock-out mice as negative controls to establish antibody specificity, we show that these relatively small amounts of EAAC1 protein are widely distributed in somata and dendrites of all hippocampal neurons. These findings raise new questions about how so few transporters can influence the activation of NMDA receptors at excitatory synapses.