Skip to Content
Merck
  • Application of MS Transport Assays to the Four Human γ-Aminobutyric Acid Transporters.

Application of MS Transport Assays to the Four Human γ-Aminobutyric Acid Transporters.

ChemMedChem (2015-07-30)
Sebastian Schmitt, Georg Höfner, Klaus T Wanner
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Hypotaurine, ≥98% (TLC)
Sigma-Aldrich
Calcium chloride
Sigma-Aldrich
Calcium chloride solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
Quinidine sulfate salt dihydrate
Sigma-Aldrich
L-Glutamine
Sigma-Aldrich
Calcium chloride, AnhydroBeads, −10 mesh, ≥99.99% trace metals basis
Sigma-Aldrich
Calcium chloride, anhydrous, powder, 99.99% trace metals basis
Sigma-Aldrich
Calcium chloride, AnhydroBeads, −10 mesh, ≥99.9% trace metals basis
Sigma-Aldrich
L-Glutamine, meets USP testing specifications, suitable for cell culture, 99.0-101.0%, from non-animal source
Sigma-Aldrich
L-Glutamine, γ-irradiated, BioXtra, suitable for cell culture
Sigma-Aldrich
L-Glutamine
SAFC
Taurine
SAFC
L-Glutamine
Sigma-Aldrich
Taurine, ≥99%
Sigma-Aldrich
Calcium chloride, anhydrous, BioReagent, suitable for insect cell culture, suitable for plant cell culture, ≥96.0%
Sigma-Aldrich
Betaine hydrochloride, ≥99%
Sigma-Aldrich
Taurine, suitable for cell culture, meets USP testing specifications
Sigma-Aldrich
L-Glutamine, BioUltra, ≥99.5% (NT)
Sigma-Aldrich
Taurine, BioUltra, ≥99.5% (T)