title: MS transport assays for γ-aminobutyric acid transporters--an efficient alternative for radiometric assays.
abstract: Transport assays for neurotransmitters based on radiolabeled substrates are widely spread and often indispensable in basic research and the drug development process, although the use of radioisotopes is inherently coupled to issues concerning radioactive waste and safety precautions. To overcome these disadvantages, we developed mass spectrometry (MS)-based transport assays for γ-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system (CNS). These "MS Transport Assays" provide all capabilities of [(3)H]GABA transport assays and therefore represent the first substitute for the latter. The performance of our approach is demonstrated for GAT1, the most important GABA transporter (GAT) subtype. As GABA is endogenously present in COS-7 cells employed as hGAT1 expression system, ((2)H6)GABA was used as a substrate to differentiate transported from endogenous GABA. To record transported ((2)H6)GABA, a highly sensitive, short, robust, and reliable HILIC-ESI-MS/MS quantification method using ((2)H2)GABA as an internal standard was developed and validated according to the Center for Drug Evaluation and Research (CDER) guidelines. Based on this LC-MS quantification, a setup to characterize hGAT1 mediated ((2)H6)GABA transport in a 96-well format was established, that enables automated processing and avoids any sample preparation. The K(m) value for ((2)H6)GABA determined for hGAT1 is in excellent agreement with results obtained from [(3)H]GABA uptake assays. In addition, the established assay format enables efficient determination of the inhibitory potency of GAT1 inhibitors, is capable of identifying those inhibitors transported as substrates, and furthermore allows characterization of efflux. The approach described here combines the strengths of LC-MS/MS with the high efficiency of transport assays based on radiolabeled substrates and is applicable to all GABA transporter subtypes.
authors.affiliation: Department of Pharmacy-Center for Drug Research, Ludwig-Maximilians-Universität München , Butenandtstraße 7, 81377 München, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Schmitt
authors.forename: Georg
authors.initials: G
authors.lastname: Höfner
authors.forename: Klaus T
authors.initials: KT
authors.lastname: Wanner
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D002853:Chromatography, Liquid; D050485:GABA Plasma Membrane Transport Proteins; D011874:Radiometry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D050485:GABA Plasma Membrane Transport Proteins