title: Application of MS Transport Assays to the Four Human γ-Aminobutyric Acid Transporters.
abstract: γ-Aminobutyric acid (GABA) transporters (GATs) are promising drug targets for various diseases associated with imbalances in GABAergic neurotransmission. For the development of new drugs or pharmacological tools addressing GATs, screening techniques to identify new inhibitors and to characterize their potency at each GAT subtype are indispensable. By now, the technique by far dominating is based on radiolabeled GABA. We recently described "MS Transport Assays" for hGAT-1 by employing ((2) H6 )GABA as the substrate. In the present study, we applied this approach to all four human GAT subtypes and determined the KM values for GAT-mediated transport of ((2) H6 )GABA at each subtype. Furthermore, a comprehensive set of GAT inhibitors reflecting the whole range of potency and subtype selectivity known so far was evaluated for their potency. The comparison of pIC50 values obtained in conventional [(3) H]GABA uptake assays with those obtained in MS Transport Assays indicated the reliability of the latter. The MS Transport Assays enable a throughput similar to that of conventional radiometric transport assays performed in a 96-well format but avoid the use of radiolabeled substrates.
authors.affiliation: Department für Pharmazie - Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 München (Germany).
authors.forename: Sebastian
authors.initials: S
authors.lastname: Schmitt
authors.affiliation: Department für Pharmazie - Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 München (Germany).
authors.forename: Georg
authors.initials: G
authors.lastname: Höfner
authors.affiliation: Department für Pharmazie - Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 München (Germany). klaus.wanner@cup.uni-muenchen.de.
authors.forename: Klaus T
authors.initials: KT
authors.lastname: Wanner
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D004353:Drug Evaluation, Preclinical / Q000379:methods; D050485:GABA Plasma Membrane Transport Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D058805:GABA Uptake Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D058990:Molecular Targeted Therapy / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D057188:Workflow; D005680:gamma-Aminobutyric Acid / Q000378:metabolism / Q000493:pharmacokinetics
chemical_list: D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D058805:GABA Uptake Inhibitors; C493413:SLC6A1 protein, human; C493416:SLC6A11 protein, human; C493418:SLC6A13 protein, human; C072207:betaine plasma membrane transport proteins; D005680:gamma-Aminobutyric Acid
keywords: competitive transport assays; human GABA transporters; mass spectrometry; neurotransmitters; subtype selectivity