Saturation Assays for mGAT1 73211 GABA Transporters

g-Amino butyric acid (GABA) is the most important inhibitory neurotransmitter in the central nervous system. In the last three decades, GABAergic neuro-

transmission was linked, directly or indirectly, to a variety of neuro-pathological and psychiatric medical conditions, e.g. epilepsy, Huntington's chorea, Parkinson, Tardive dyskinesia, schizophrenia, anxiety, depression and other behavioral disorders. The GABA transporters (GATs) that remove the neurotransmitter from the synaptic cleft after its release are very interesting targets for the development of new agents for the indications listed above [81]. Inhibitors of these transporters can extend the presence of GABA in the synaptic cleft and therefore increase the inhibitory effect of the neurotransmitter. For the GABA transporters that are a part of the superfamily of Na+/Cl~-dependent transporters four subtypes are known. Among them the transporter subtype most frequently found in the brain is GAT1, an already validated target in the search for anticonvulsants [82-85]. A successful example is the agent tiagabine, an effective and selective inhibitor of GAT1 which has already been introduced in the therapy of epilepsy in the form of the drug Gabitril [81-83, 85-88].

For in vitro screening of new ligands at GABA transporters uptake assays are generally used which measure the uptake of [3H]GABA into whole cells or synaptosomes [89-92]. However, this type of assay has significant practical drawbacks. It is necessary to employ whole cells or synaptosomes that have to stay intact during the entire course of the experiment, i.e. also during the separation step, since the breakdown of the cell membrane or the synaptosome would cause a loss of substrate ([3H]GABA). In contrast, conventional binding assays which determine not the functionality but the affinity for the GABA transporter have the advantage that membrane fractions are sufficient for these experiments [93, 94].

In 1990, Braestrup and coworkers reported the first radioligand binding assay for GABA transporters [95-97]. The radioligand they employed in their experiments was [3H]tiagabine. Unfortunately, neither [3H]tiagabine nor other radioligands for binding assays to GAT1 are readily commercially available. There is, however, no shortage of affine and selective ligands for GAT1 [98]. This is another intriguing fact highlighting the advantages of MS binding assays which can be conducted without a labeled marker.

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