M Ruiz1, H Egal, V Sarthy, X Qian, H K Sarkar. 1. Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.
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.
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.