Pharmacological Characterization of [<sup>3</sup>H]ATPCA
as a Substrate for Studying the Functional Role of the Betaine/GABA
Transporter 1 and the Creatine Transporter
The
betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT1)
is one of the four GABA transporters (GATs) involved in the termination
of GABAergic neurotransmission. Although suggested to be implicated
in seizure management, the exact functional importance of BGT1 in
the brain is still elusive. This is partly owing to the lack of potent
and selective pharmacological tool compounds that can be used to probe
its function. We previously reported the identification of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic
acid (ATPCA), a selective substrate for BGT1 over GAT1/GAT3, but also
an agonist for GABA<sub>A</sub> receptors. With the aim of providing
new functional insight into BGT1, we here present the synthesis and
pharmacological characterization of the tritiated analogue, [<sup>3</sup>H]ATPCA. Using traditional uptake assays at recombinant transporters
expressed in cell lines, [<sup>3</sup>H]ATPCA displayed a striking
selectivity for BGT1 among the four GATs (<i>K</i><sub>m</sub> and <i>V</i><sub>max</sub> values of 21 μM and 3.6
nmol ATPCA/(min × mg protein), respectively), but was also found
to be a substrate for the creatine transporter (CreaT). In experiments
with mouse cortical cell cultures, we observed a Na<sup>+</sup>-dependent
[<sup>3</sup>H]ATPCA uptake in neurons, but not in astrocytes. The
neuronal uptake could be inhibited by GABA, ATPCA, and a noncompetitive
BGT1-selective inhibitor, indicating functional BGT1 in neurons. In
conclusion, we report [<sup>3</sup>H]ATPCA as a novel radioactive
substrate for both BGT1 and CreaT. The dual activity of the radioligand
makes it most suitable for use in recombinant studies