Development of a scintillation proximity assay for analysis of Na+/Cl- -dependent neurotransmitter transporter activity (vol 321, pg 31, 2003)

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 [14C]glycine was time dependent and saturable with a Michaelis–Menten constant (Km) of 27±3μM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [14C]glycine uptake with expected IC50 values of 37.5±4.6μM, 2.8±0.6nM, and 6.9±0.9nM, respectively. The [14C]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 [14C]taurine uptake in JAR cells. Taurine transport was of high affinity with a Km of 10.2±1.7μM and fully inhibited by ALX-5407 (IC50=522 ±83nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter inhibitors.