TY - JOUR
T1 - Pharmacological Characterization of [3H]ATPCA as a Substrate for Studying the Functional Role of the Betaine/GABA Transporter 1 and the Creatine Transporter
AU - Al-Khawaja, Anas
AU - Haugaard, Anne S
AU - Marek, Ales
AU - Löffler, Rebekka
AU - Thiesen, Louise
AU - Santiveri, Monica
AU - Damgaard, Maria
AU - Bundgaard, Christoffer
AU - Frølund, Bente
AU - Wellendorph, Petrine
PY - 2018/3/21
Y1 - 2018/3/21
N2 - 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, [ 3 H]ATPCA. Using traditional uptake assays at recombinant transporters expressed in cell lines, [ 3 H]ATPCA displayed a striking selectivity for BGT1 among the four GATs (K m and V 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 [ 3 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 [ 3 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.
AB - 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, [ 3 H]ATPCA. Using traditional uptake assays at recombinant transporters expressed in cell lines, [ 3 H]ATPCA displayed a striking selectivity for BGT1 among the four GATs (K m and V 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 [ 3 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 [ 3 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.
KW - Journal Article
U2 - 10.1021/acschemneuro.7b00351
DO - 10.1021/acschemneuro.7b00351
M3 - Journal article
C2 - 29131576
SN - 1948-7193
VL - 9
SP - 545
EP - 554
JO - A C S Chemical Neuroscience
JF - A C S Chemical Neuroscience
IS - 3
ER -