Jursky F, Tamura S, Tamura A, Mandiyan S, Nelson H, Nelson N
Roche Institute of Molecular Biology, Nutley, NJ 07110.
J Exp Biol. 1994 Nov;196:283-95. doi: 10.1242/jeb.196.1.283.
We studied four different cDNAs encoding GABA transporters and three different cDNAs encoding glycine transporters in mouse and rat brains. A genomic clone of two of the glycine transporters (GLYT1a and GLYT1b) revealed that they derive from differential splicing of a single gene. The third glycine transporter (GLYT2) is encoded by a separate gene. Antibodies were raised against seven of these neurotransmitter transporters and their cytochemical localization in the mouse brain was studied. In general, we observed a deviation from the classical separation of neuronal and glial transporters. It seems that each of the neurotransmitter transporters is present in specific places in the brain and is expressed in a different way in very specific areas. For example, the GABA transporter GAT4, which also transports beta-alanine, was localized to neurons. However, GAT1, which is specific for GABA, was localized not only to neurons but also to glial cells. The recently discovered glycine transporter GLYT2 was of particular interest because of its deviation from the general structure by a very extended N terminus containing multiple potential phosphorylation sites. Western analysis and immunocytochemistry in frozen sections of mouse brain demonstrated a clear caudal-rostral gradient of GLYT2 distribution, with massive accumulation in the spinal cord and brainstem and less in the cerebellum. Its distribution is typically neuronal and it is present in processes with varicosities. A correlation as observed between the pattern we obtained and that observed previously from strychnine binding studies. The results indicate that GLYT2 is involved in the termination of glycine neurotransmission at the classical inhibitory system in the hindbrain. The availability of four different GABA transporters made it possible to look for specific binding sites upon the neurotransmitter transporters. An extensive program of site-directed mutagenesis led us to identify a potential neurotransmitter binding site on the GABA transporters.
我们研究了小鼠和大鼠脑中四种编码GABA转运体的不同cDNA以及三种编码甘氨酸转运体的不同cDNA。两种甘氨酸转运体（GLYT1a和GLYT1b）的基因组克隆显示它们源自单个基因的可变剪接。第三种甘氨酸转运体（GLYT2）由一个单独的基因编码。针对其中七种神经递质转运体产生了抗体，并研究了它们在小鼠脑中的细胞化学定位。总体而言，我们观察到与神经元和胶质细胞转运体的经典区分存在偏差。似乎每种神经递质转运体都存在于大脑的特定部位，并在非常特定的区域以不同的方式表达。例如，也转运β-丙氨酸的GABA转运体GAT4定位于神经元。然而，对GABA具有特异性的GAT1不仅定位于神经元，还定位于胶质细胞。最近发现的甘氨酸转运体GLYT2因其N末端非常长且含有多个潜在磷酸化位点而与一般结构不同，因而特别受关注。小鼠脑冰冻切片的蛋白质免疫印迹分析和免疫细胞化学显示GLYT2分布存在明显的尾-头梯度，在脊髓和脑干中大量积累，而在小脑中较少。其分布典型地为神经元性，并且存在于有曲张体的突起中。我们获得的模式与先前从士的宁结合研究中观察到的模式之间存在相关性。结果表明GLYT2参与后脑经典抑制系统中甘氨酸神经传递的终止。四种不同GABA转运体的可得性使得寻找神经递质转运体上的特异性结合位点成为可能。广泛的定点诱变程序使我们能够在GABA转运体上鉴定出一个潜在的神经递质结合位点。
