Pharmacological Characterization of [3H]ATPCA as a Substrate for Studying the Functional Role of the Betaine/GABA Transporter 1 and the Creatine Transporter

Anas Al-Khawaja; Anne S Haugaard; Ales Marek; Rebekka Löffler; Louise Thiesen; Monica Santiveri; Maria Damgaard; Christoffer Bundgaard; Bente Frølund; Petrine Wellendorph

doi:10.1021/acschemneuro.7b00351

Pharmacological Characterization of [³H]ATPCA as a Substrate for Studying the Functional Role of the Betaine/GABA Transporter 1 and the Creatine Transporter

Anas Al-Khawaja, Anne S Haugaard, Ales Marek, Rebekka Löffler, Louise Thiesen, Monica Santiveri, Maria Damgaard, Christoffer Bundgaard, Bente Frølund, Petrine Wellendorph

5 Citations (Scopus)

Abstract

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.

Original language	English
Journal	A C S Chemical Neuroscience
Volume	9
Issue number	3
Pages (from-to)	545–554
ISSN	1948-7193
DOIs	https://doi.org/10.1021/acschemneuro.7b00351
Publication status	Published - 21 Mar 2018

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Journal Article

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10.1021/acschemneuro.7b00351

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Al-Khawaja, A., Haugaard, A. S., Marek, A., Löffler, R., Thiesen, L., Santiveri, M., Damgaard, M., Bundgaard, C., Frølund, B., & Wellendorph, P. (2018). Pharmacological Characterization of [³H]ATPCA as a Substrate for Studying the Functional Role of the Betaine/GABA Transporter 1 and the Creatine Transporter. A C S Chemical Neuroscience, 9(3), 545–554. https://doi.org/10.1021/acschemneuro.7b00351

Pharmacological Characterization of [³H]ATPCA as a Substrate for Studying the Functional Role of the Betaine/GABA Transporter 1 and the Creatine Transporter. / Al-Khawaja, Anas; Haugaard, Anne S; Marek, Ales et al.

In: A C S Chemical Neuroscience, Vol. 9, No. 3, 21.03.2018, p. 545–554.

Research output: Contribution to journal › Journal article › Research › peer-review

Al-Khawaja, A, Haugaard, AS, Marek, A, Löffler, R, Thiesen, L, Santiveri, M, Damgaard, M, Bundgaard, C, Frølund, B & Wellendorph, P 2018, 'Pharmacological Characterization of [³H]ATPCA as a Substrate for Studying the Functional Role of the Betaine/GABA Transporter 1 and the Creatine Transporter', A C S Chemical Neuroscience, vol. 9, no. 3, pp. 545–554. https://doi.org/10.1021/acschemneuro.7b00351

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abstract = " 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. ",

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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

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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.

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