In this study, the γ-aminobutyric acid (GABA) transporter at the blood-brain barrier (BBB) was identified by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunostaining analysis, and the transport mechanism was characterized using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB) as an in vitro model of the BBB. γ-Aminobutyric acid transport was studied by the cellular uptake of [3H]GABA. [3H]GABA uptake by TM-BBB cells was Na+-, Cl--, and concentration-dependent. The corresponding Michaelis-Menten constant was 679 ± 80 μmol/L and the maximal uptake rate was 4,790 ± 494 pmol/(mg protein · 5 minutes). [3H]GABA uptake by TM-BBB cells was significantly inhibited by betaine, β-alanine, nipecotic acid, taurine, and quinidine, whereas probenecid, L-proline, creatine, and glycine had no effect. This type of inhibition is consistent with the predominant involvement of the GAT2/BGT-1 transporter in TM-BBB cells. RT-PCR analysis showed that GAT2/BGT-1 mRNA was expressed in TM-BBB cells, whereas Western blot analysis showed that TM-BBB cells and mouse brain capillaries express GAT2/BGT-1 protein. Moreover, confocal immunofluorescent microscopy of dual-labeled mouse brain sections demonstrated the colocalization of GAT2/BGT-1 and P-glycoprotein, a BBB-specific marker, on brain capillaries labeled with anti-GAT2/BGT-1 antibody and anti-P-glycoprotein antibody, respectively. These results are evidence that GAT2/BGT-1 is expressed at the BBB and is involved in GABA transport across the BBB.
ASJC Scopus subject areas
- Clinical Neurology
- Cardiology and Cardiovascular Medicine