Pharmacological Characterization of [³H]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_A receptors. With the aim of providing new functional insight into BGT1, we here present the synthesis and pharmacological characterization of the tritiated analogue, [³H]­ATPCA. Using traditional uptake assays at recombinant transporters expressed in cell lines, [³H]­ATPCA displayed a striking selectivity for BGT1 among the four GATs (<i>K</i>_m and <i>V</i>_max 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⁺-dependent [³H]­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 [³H]­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–Function Evaluation of Imidazopyridine Derivatives Selective for δ‑Subunit-Containing γ‑Aminobutyric Acid Type A (GABA_A) Receptors

δ-Selective compounds 1 and 2 (DS1, compound <b>22</b>; DS2, compound <b>16</b>) were introduced as functionally selective modulators of δ-containing GABA type A receptors (GABA_AR). In our hands, [³H]­EBOB-binding experiments with recombinant GABA_AR and compound <b>22</b> showed no proof of δ-selectivity, although there was a minimally higher preference for the α4β3δ and α6β2/3δ receptors with respect to potency. In order to delineate the structural determinants of δ preferences, we synthesized 25 derivatives of DS1 and DS2, and investigated their structure–activity relationships (SAR). Four of our derivatives showed selectivity for α6β3δ receptors (<b>29</b>, <b>38</b>, <b>39</b>, and <b>41</b>). For all of them, the major factors that distinguished them from compound <b>22</b> were variations at the <i>para</i>-positions of their benzamide groups. However, two compounds (<b>29</b> and <b>39</b>), when tested in the presence of GABA, revealed effects at several additional GABA_AR. The newly synthesized compounds will still serve as useful tools to investigate α6β3δ receptors