First Demonstration of a Functional Role for Central Nervous System Betaine/γ-Aminobutyric Acid Transporter (mGAT2) Based on Synergistic Anticonvulsant Action among Inhibitors of mGAT1 and mGAT2

• Published in: The Journal of pharmacology and experimental therapeutics Vol. 312; no. 2; p. 866
• Main Authors: H. Steve White, William P. Watson, Suzanne L. Hansen, Scott Slough, Jens Perregaard, Alan Sarup, Tina Bolvig, Gitte Petersen, Orla M. Larsson, Rasmus P. Clausen, Bente Frølund, Erik Falch, Povl Krogsgaard-Larsen, Arne Schousboe
• Format: Journal Article
• Language: English
• Published: American Society for Pharmacology and Experimental Therapeutics 01.02.2005
• ISSN: 0022-3565; 1521-0103
• DOI: 10.1124/jpet.104.068825

In a recent study, EF1502 [ N -[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo [ d ]isoxazol-3-ol], which is an N -substituted analog of the GAT1-selective GABA uptake inhibitor exo -THPO (4-amino-4,5,6,7-tetrahydrobenzo[ d ]isoxazol-3-ol), was found to inhibit GABA transport mediated by both GAT1 and GAT2 in human embryonic kidney (HEK) cells expressing the mouse GABA transporters GAT1 to 4 (mGAT1–4). In the present study, EF1502 was found to possess a broad-spectrum anticonvulsant profile in animal models of generalized and partial epilepsy. When EF1502 was tested in combination with the clinically effective GAT1-selective inhibitor tiagabine [( R )- N -[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid] or LU-32-176B [ N -[4,4-bis(4-fluorophenyl)-butyl]-3-hydroxy-4-amino-4,5,6,7-tetrahydrobenzo[ d ]isoxazol-3-ol], another GAT1-selective N -substituted analog of exo -THPO, a synergistic rather than additive anticonvulsant interaction was observed in the Frings audiogenic seizure-susceptible mouse and the pentylenetetrazol seizure threshold test. In contrast, combination of the two mGAT1-selective inhibitors, tiagabine and LU-32-176B, resulted in only an additive anticonvulsant effect. Importantly, the combination of EF1502 and tiagabine did not result in a greater than additive effect in the rotarod behavioral impairment test. In subsequent in vitro studies conducted in HEK-293 cells expressing the cloned mouse GAT transporters mGAT1 and mGAT2, EF1502 was found to noncompetitively inhibit both mGAT1 and the betaine/GABA transporter mGAT2 ( K i of 4 and 5 μM, respectively). Furthermore, in a GABA release study conducted in neocortical neurons, EF1502 did not act as a substrate for the GABA carrier. Collectively, these findings support a functional role for mGAT2 in the control of neuronal excitability and suggest a possible utility for mGAT2-selective inhibitors in the treatment of epilepsy.