2 research outputs found
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
Structure–Activity Relationship, Pharmacological Characterization, and Molecular Modeling of Noncompetitive Inhibitors of the Betaine/γ-Aminobutyric Acid Transporter 1 (BGT1)
<i>N</i>-(1-Benzyl-4-piperidinyl)-2,4-dichlorobenzamide <b>5</b> (BPDBA) is a noncompetitive inhibitor of the betaine/GABA
transporter 1 (BGT1). We here report the synthesis and structure–activity
relationship of 71 analogues. We identify <b>26m</b> as a more
soluble 2,4-Cl substituted 3-pyridine analogue with retained BGT1
activity and an improved off-target profile compared to <b>5</b>. We performed radioligand-based uptake studies at chimeric constructs
between BGT1 and GAT3, experiments with site-directed mutated transporters,
and computational docking in a BGT1 homology model based on the newly
determined X-ray crystal structure of the human serotonin transporter
(hSERT). On the basis of these experiments, we propose a binding mode
involving residues within TM10 in an allosteric site in BGT1 that
corresponds to the allosteric binding pocket revealed by the hSERT
crystal structure. Our study provides first insights into a proposed
allosteric binding pocket in BGT1, which accommodates the binding
site for a series of novel noncompetitive inhibitors