Role of TauT in GABA Transport at the Inner Blood-Retinal Barrier

Abstract

Purpose:: γ-Aminobutyric acid (GABA) plays a key role as an inhibitory neurotransmitter in the retina. The regulation of GABA concentration in the retina is still poorly understood. The purpose of this study was to elucidate the GABA transport system at the inner blood-retinal barrier (inner BRB).

Methods:: Conditionally immortalized rat retinal endothelial cell line (TR-iBRB cells) was cultured in DMEM supplemented with 10% FBS at 33°C in humidified atmosphere of 5% CO₂/air. Identification and localization of taurine transporter (TauT) were determined by RT-PCR and immunoblot analyses. Rat TauT cDNA was amplified by RT-PCR from total RNA of TR-iBRB cells, and ligated into pcDNA4/HisC. Constructed pcDNA4/HisC/TauT was transfected into Hela cells using Lipofectamine 2000. The uptake studies were performed at 37°C by applying radiolabeled amino acids to cells in the presence or absence of inhibitors.

Results:: The uptake of [³H]GABA by TR-iBRB cells was concentration-dependent with the K_m of 2.0 mM. This process was more potently inhibited by substrates of taurine transporter (TauT), taurine and ß-alanine, than those of GABA transporters (GATs), GABA and betaine. In the presence of taurine, there was competitive inhibition with a K_i of 78 µM. [³H]Taurine uptake was saturable with a K_m of 38 µM, and it exhibited competitive inhibition with a K_i of 1.8 mM in the presence of GABA. The mutual inhibition between GABA and taurine and type of inhibition suggest that TauT mediates GABA uptake in TR-iBRB cells. RT-PCR and immunoblot analyses demonstrated that TauT is expressed in TR-iBRB and primary cultured human retinal endothelial cells. The uptake rate of [³H]GABA in Hela cells-expressing Xpress-tagged rat TauT was 4.7-fold greater than that of mock cells, and the TauT-mediated uptake of [³H]GABA was Na⁺-, Cl^--, and concentration- dependent with a K_m of 1.5 mM. Moreover, the TauT-mediated uptake of [³H]GABA in Hela cells was strongly inhibited by TauT substrates, taurine and ß-alanine.

Conclusions:: TauT-mediated GABA transport appears to be present at the inner BRB.

Keywords: retina: neurochemistry • taurine • retinal culture