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

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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

Abstract

Purpose. To isolate a cDNA clone encoding a high-affinity γ-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 K(m) and V(max) for the GABA uptake were 8.3 μM 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 μM, respectively. However, β-alanine was unable to inhibit the induced GABA uptake significantly (IC50 ≃ 2,500 μM). 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 Muller 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.

Original languageEnglish (US)
Pages (from-to)4039-4048
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume35
Issue number12
StatePublished - 1994

Keywords

  • GABA transporter
  • cloning and expression
  • in situ localization
  • retinal neurotransmitter

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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