Cloning, expression, and localization of a mouse retinal gamma-aminobutyric acid transporter.
OBJECTIVE
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.
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.
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Ruiz
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Sarthy
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D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D015342:DNA Probes; D018076:DNA, Complementary / Q000032:analysis / Q000302:isolation & purification; D050485:GABA Plasma Membrane Transport Proteins; D015870:Gene Expression; D017403:In Situ Hybridization; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D026901:Membrane Transport Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D009865:Oocytes / Q000378:metabolism; D027361:Organic Anion Transporters; D012333:RNA, Messenger / Q000302:isolation & purification; D012160:Retina / Q000378:metabolism*; D014981:Xenopus / Q000235:genetics; D005680:gamma-Aminobutyric Acid / Q000235:genetics* / Q000378:metabolism*
D002352:Carrier Proteins; D015342:DNA Probes; D018076:DNA, Complementary; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D012333:RNA, Messenger; C493414:Slc6a1 protein, mouse; C493415:Slc6a1 protein, rat; D005680:gamma-Aminobutyric Acid