Characterization and distribution of the neuronal glutamate transporter EAAC1 in rat brain.
Characterization and distribution of the neuronal glutamate transporter EAAC1 in rat brain.
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DOI：
10.1152/ajpcell.1996.270.1.c67
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发表时间：
1996
期刊：
影响因子：
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通讯作者：
M. Velaz-Faircloth;T. S. McGraw;M. S. alandro;R. Fremeau;M. Kilberg;K. Anderson
中科院分区：
文献类型：
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作者：
M. Velaz-Faircloth;T. S. McGraw;M. S. alandro;R. Fremeau;M. Kilberg;K. Anderson
谷氨酸和其他相关兴奋性氨基酸（EAA）的细胞外浓度是由位于突触前末梢或相邻星形胶质细胞过程的转运蛋白的作用调节。最近的分子学进展已经克隆了三个独立的编码Na（+）依赖性谷氨酸转运蛋白的cDNA;其中两个被认为主要来源于胶质细胞（GLAST和GLT-1），第三个（EAAC 1）定位于脑和其他非神经组织中的神经元。EAAC 1 cDNA最初是从兔小肠克隆的（13）。在这项研究中，我们报告了从大鼠大脑中分离和鉴定同源克隆的情况。北方印迹杂交显示，EAAC 1 mRNA在大鼠脑和肾脏中的高水平，在心脏、肺和骨骼肌中的低水平。EAAC 1在HeLa细胞中的瞬时表达导致Na（+）依赖的高亲和力L-[3 H]谷氨酸和D-[3 H]天冬氨酸转运的增加。EAAC 1的药理学特征与所报道的兔和人EAAC 1同源物非常相似。D-和L-苏型-β-羟基天冬氨酸和L-反式-吡咯烷-2，4-二羧酸可有效抑制转运活性。二氢红藻氨酸和L-α-aminoadipate没有抑制运输浓度低于1 mM。寡核苷酸cDNA探针（45聚体）的构建和标记与35 S-ATP的电影和乳液为基础的原位杂交大鼠大脑。EAAC 1 mRNA在小脑颗粒细胞层、海马、上级丘和新皮质中密度最高。用甲酚紫进行乳液浸渍和复染的切片显示EAAC 1标记仅定位于神经元细胞体上，包括一些非谷氨酸能神经元，如脊髓腹角细胞。
The extracellular concentration of glutamate and other related excitatory amino acids (EAA) is regulated by the action of transporter proteins located on either presynaptic terminals or adjacent astroglial processes. Recent molecular advances have led to the cloning of three separate cDNAs encoding for Na(+)-dependent glutamate transporters; two are thought to be primarily glial in origin (GLAST and GLT-1) and the third (EAAC1) is localized to neurons in the brain and other nonneural tissues. An EAAC1 cDNA was initially cloned from rabbit small intestine (13). In this study, we report isolation and characterization of the homologous clone from rat brain. Northern blot hybridization revealed high levels of EAAC1 mRNA in rat brain and kidney and low levels in heart, lung, and skeletal muscle. Transient expression of EAAC1 in HeLa cells resulted in an increase in Na(+)-dependent high-affinity L-[3H]glutamate and D-[3H]aspartate transport. The pharmacological profile of EAAC1 was very similar to that reported for the rabbit and human EAAC1 homologues. Transport activity was potently inhibited by D- and L-threo-beta-hydroxyaspartate and L-trans-pyrrolodine-2,4-dicarboxylate. Dihydrokainate and L-alpha-aminoadipate did not inhibit transport at concentrations below 1 mM. Oligonucleotide cDNA probes (45-mer) were constructed and labeled with 35S-ATP for film- and emulsion-based in situ hybridization of rat brain. EAAC1 mRNA had the highest density in the cerebellar granule cell layer, hippocampus, superior colliculus, and neocortex. Sections that were emulsion-dipped and counterstained with cresyl violet revealed EAAC1 labeling localized exclusively over neuronal cell bodies, including some nonglutamatergic neurons such as spinal cord ventral horn cells.