小鼠视网膜γ-氨基丁酸转运体的克隆、表达及定位

Cloning, expression, and localization of a mouse retinal gamma-aminobutyric acid transporter.

作者信息

Ruiz M, Egal H, Sarthy V, Qian X, Sarkar H K

机构信息

Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.

出版信息

Invest Ophthalmol Vis Sci. 1994 Nov;35(12):4039-48.

PMID:7960586

Abstract

PURPOSE

To isolate a cDNA clone encoding a high-affinity gamma-aminobutyric acid (GABA) transporter from mouse retina, to examine its biochemical and pharmacologic properties, and to determine the sites of its mRNA expression in retinal cells.

METHODS

A mouse retinal cDNA library was screened using a fragment of a rat brain GABA transporter (GAT-1) cDNA as a probe. One homologous clone, mouse retinal GAT-1, was chosen for further characterization. RNA transcribed from mouse retinal GAT-1 was microinjected into Xenopus oocytes, and pharmacologic properties of the expressed transporter were determined. Sites of mouse retinal GAT-1 mRNA expression were examined by in situ hybridization.

RESULTS

The protein sequence deduced from the DNA sequence of mouse retinal GAT-1 cDNA was virtually identical to that of the rat and the mouse brain GAT-1. RNA transcribed from this clone induced a [3H]-GABA uptake activity in microinjected Xenopus oocytes that was both sodium and chloride dependent. The apparent Km and Vmax for the GABA uptake were 8.3 microM and 40.0 pmol/egg per hour, respectively. The mouse retinal GAT-1 induced GABA uptake was inhibited by L-diaminobutyric acid, guvacine, cis-4-hydroxynipecotic acid, nipecotic acid, and 4,5,6,7-tetrahydroisoxazolo [4,5c]-pyridin-3-ol with IC50 values of 320, 79, 71, 7.1, and 200 microM, respectively. However, beta-alanine was unable to inhibit the induced GABA uptake significantly (IC50 approximately 2,500 microM). In situ hybridization studies showed that mouse retinal GAT-1 mRNA was present in a subpopulation of amacrine, interplexiform, and displaced amacrine cells. Hybridization signal in the Müller cells was significantly lower, and GAT-1 transcripts were not detected in the bipolar, horizontal, or photoreceptor cells of mouse retina.

CONCLUSIONS

The mouse retinal GAT-1 cDNA encodes a Na(+)-dependent, high-affinity GABA transporter that is mainly expressed in a subset of mouse retinal inter neurons.

摘要

目的

从小鼠视网膜中分离出编码高亲和力γ-氨基丁酸（GABA）转运体的cDNA克隆，研究其生化和药理学特性，并确定其mRNA在视网膜细胞中的表达位点。

方法

以大鼠脑GABA转运体（GAT-1）cDNA片段为探针，筛选小鼠视网膜cDNA文库。选择一个同源克隆，即小鼠视网膜GAT-1，进行进一步鉴定。将从小鼠视网膜GAT-1转录的RNA显微注射到非洲爪蟾卵母细胞中，测定所表达转运体的药理学特性。通过原位杂交检测小鼠视网膜GAT-1 mRNA的表达位点。

结果

从小鼠视网膜GAT-1 cDNA的DNA序列推导的蛋白质序列与大鼠和小鼠脑GAT-1的序列几乎相同。从该克隆转录的RNA在显微注射的非洲爪蟾卵母细胞中诱导出一种[3H]-GABA摄取活性，该活性依赖于钠和氯。GABA摄取的表观Km和Vmax分别为8.3 μM和40.0 pmol/卵/小时。L-二氨基丁酸、胍氨酸、顺式-4-羟基尼克酸、尼克酸和4,5,6,7-四氢异恶唑并[4,5c]-吡啶-3-醇可抑制小鼠视网膜GAT-1诱导的GABA摄取，IC50值分别为320、79、71、7.1和200 μM。然而，β-丙氨酸不能显著抑制诱导的GABA摄取（IC50约为2500 μM）。原位杂交研究表明，小鼠视网膜GAT-1 mRNA存在于无长突细胞、网间细胞和移位无长突细胞亚群中。在穆勒细胞中的杂交信号明显较低，在小鼠视网膜的双极细胞、水平细胞或光感受器细胞中未检测到GAT-1转录本。