The Four Human γ-Aminobutyric Acid (GABA) Transporters: Pharmacological Characterization and Validation of a Highly Efficient Screening Assay

Abstract

The neurotransmission mediated by gamma-aminobutyric acid (GABA) in the mammalian brain is terminated by a family of four GABA transporters (GATs). Inhibition of GATs is currently used in the treatment of epilepsy and these proteins are generally considered as important drug targets. In this study, we perform the first elaborate pharmacological characterization of all four human GAT subtypes. We conduct the experiments in parallel in a [3H]GABA uptake assay using 14 standard GAT substrates and inhibitors. This setup enables direct comparison of the absolute values of inhibitory activities of the compounds between the different GAT subtypes. The results are overall in agreement with data reported by other groups for the orthologous murine GATs. However, there do seem to be some minor variations among species. In contrast to the several subtype selective ligands identified for the GAT-1 subtype, no subtype selective ligands have been reported for the three remaining GATs. Given the potential therapeutic relevance of the individual GAT subtypes, a search for novel structures displaying selectivities for specific GAT subtypes is important. In this study, we validate our [3H]GABA uptake assay for use in high throughput screening. We find that the assay is categorized by high Z'-factors (Z' > 0.5) for all four GAT subtypes, demonstrating that the assay is excellent for a high throughput screen. This [3H]GABA uptake assay therefore enables future high throughput screening of compound libraries at the four human GATs.

MS Transport Assays for γ-Aminobutyric Acid Transporters—An Efficient Alternative for Radiometric Assays

“…This ( 2 H 6 )GABA transport could not effectively be blocked by 10 μM NO 711, which is a potent GAT1 inhibitor with an IC 50 value of ∼100 nM, but by the use of 25 mM rac-nipecotic acid (IC 50 ≈ 3.8 μM) that is characterized by a poor GAT subtype selectivity, in contrast to NO 711. 19 This transport was found to be also sodiumdependent (see Na-free uptake in Figure 2a), and may be caused by low endogenous expression of sodium-dependent transporters, the major extent of which, however, cannot be GAT1. Though nipecotic acid, as well as GABA, are often used to determine nonspecific uptake in [ 3 H]GABA uptake experiments, 17,19,20 this approach is not suitable for the present assay, as both specific and nonspecific transport would be blocked, thus falsifying specific uptake determined under these conditions.…”

(1S, 3S)-3-Amino-4-difluoromethylenyl-1-cyclopentanoic Acid (CPP-115), a Potent γ-Aminobutyric Acid Aminotransferase Inactivator for the Treatment of Cocaine Addiction

“…The plasmids encoding hGAT-1, hBGT-1, hGAT-2, and hGAT-3, 35 respectively, were transfected into tsA201 cells using PolyFect according to the protocol of the manufacturer (Qiagen, West Sussex, UK). The next day, the tsA201 cells transiently expressing each of the four human GABA transporter subtypes were split into poly-D-lysine-coated white 96-well plates (PerkinElmer).…”

SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

“…Important examples include tiagabine and 1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid (SKF89976A), which are derivatives of nipecotic acid, and 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid (NNC-711) and 1-(2-(bis(4-(trifluoromethyl)phenyl)methox)ethyl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid (CI-966), which are THE SLC6 NEUROTRANSMITTER TRANSPORTERS 605 derivatives of guvacine. Still, these compounds are some of the most potent and selective GAT1 inhibitors identified (Nielsen et al, 1991;Andersen et al, 1993;Borden et al, 1994;Dhar et al, 1996;White et al, 2002;Kragler et al, 2005;Kvist et al, 2009) (Table 4).…”

“…After the cloning of the four GAT subtypes, it was realized that guvacine and nipecotic acid have selectivity for GAT1 over GAT3 with little or no affinity for BGT-1 and GAT2 ( Thomsen et al, 1997;Liu et al, 2003;Kragler et al, 2005;Kvist et al, 2009). Although guvacine and nipecotic acid do not readily cross the blood-brain barrier (Krogsgaard-Larsen et al, 1987), lipophilic derivatives of these substrates represent the most important biologically active GAT inhibitors developed.…”

“…GAT inhibitors with activity on GAT2, GAT3, and/or BGT1 include (S)-1-(2-(tris(4-methoxyphenyl)methoxy) ethyl)-3-piperidinecarboxylic acid (SNAP-5114), which has selectivity for GAT2 and GAT3 over BGT-1 but little or no activity at GAT1 Borden, 1996;Kvist et al, 2009), and (R)-N-(4,4-bis(3-methyl-2-thienyl)-3-butenyl)-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo(d)isoxazol-3-ol (EF1502), which possesses dualacting noncompetitive inhibitory activity at the BGT-1 and GAT1 White et al, 2005) (Table 4). Assessment of these compounds in animal models of partial and generalized epilepsy indicates that BGT-1 is a potential antiepileptic drug target (White et al, 2005;Madsen et al, 2009).…”