CLONING, EXPRESSION, AND LOCALIZATION OF A MOUSE RETINAL GAMMA-AMINOBUTYRIC-ACID TRANSPORTER

Purpose. To isolate a cDNA clone encoding a high-affinity gamma-aminob utyric acid (GABA) transporter from mouse retina, to examine its bioch emical and pharmacologic properties, and to determine the sites of its mRNA expression in retinal cells. Methods. A mouse retinal cDNA libra ry 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 cho sen for further characterization. RNA transcribed from mouse retinal G AT-1 was microinjected into Xaopus oocytes, and pharmacologic properti es of the expressed transporter were determined. Sites of mouse retina l GAT-1 mRNA expression were examined by in situ hybridization. Result s. 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 b rain GAT-1. RNA transcribed from this clone induced a [H-3]-GABA uptak e activity in microinjected Xenopus oocytes that was both sodium and c hloride dependent. The apparent K, and V,, for the GABA uptake were 8. 3 mu M and 40.0 pmol/egg per hour, respectively. The mouse retinal GAT -I induced GABA uptake was inhibited by L-diaminobutyric acid, guvacin e, cis-4-hydroxynipecotic acid, nipecotic acid, and 4,5,6,7-tetrahydro isoxazolo [4,5c]-pyridin-3-ol with IC50 values of 320, 79, 71, 7.1, an d 200 mu M, respectively. However, beta-alanine was unable to inhibit the induced GABA uptake significantly (IC50 approximate to 2,500 mu M) . In situ hybridization studies showed that mouse retinal GAT-1 mRNA w as present in a subpopulation of amacrine, interplexiform, and displac ed amacrine cells. Hybridization signal in the Muller cells was signif icantly lower, and GAT-1 transcripts were not detected in the bipolar, horizontal, or photoreceptor cells of mouse retina. Conclusions. The mouse retinal GAT-I cDNA encodes a Na+-dependent, high-affinity GABA t ransporter that is mainly expressed in a subset of mouse retinal inter neurons.