Development of a scintillation proximity assay for analysis of Na+/Cl--dependent neurotransmitter transporter activity

Human placental choriocarcinoma (JAR) cells endogenously expressing glycine transporter type 1a (GlyT1a) have been cultured in 96-well scintillating microplates to develop a homogenous screening assay for the detection of GlyT1 antagonists. In these microplates uptake of [C-14]glycine was time dependent and saturable with a Michaelis-Menten constant (K-m) of 27 +/- 3 muM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [C-14]glycine uptake with expected IC50 values of 37.5 +/- 4.6 muM, 2.8 +/- 0.6 nM, and 6.9 +/- 0.9 nM, respectively. The [C-14]glycine uptake process was sensitive to membrane Na+ gradient as blockade of membrane Na+/K+-ATPase by ouabain or Na+ exchanger by benzamil-disrupted glycine accumulation in JAR cells. Glycine influx was not affected by concentration of dimethyl sulfoxide up to 2%. The versatility of this technological approach was further confirmed by the characterization of a saturable [C-14]taurine uptake in JAR cells. Taurine transport was of high affinity with a K-m of 10.2 +/- 1.7 muM and fully inhibited by ALX-5407 (IC50 = 522 +/- 83 nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter inhibitors. (C) 2003 Elsevier Inc. All rights reserved.