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

Abstract 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 [ 14 C]glycine was time dependent and saturable with a Michaelis–Menten constant ( K m ) of 27 ± 3 μM. The GlyT1 transport inhibitors sarcosine, ALX-5407, and Org-24598 were tested and shown to block [ 14 C]glycine uptake with expected IC 50 values of 37.5 ± 4.6 μM, 2.8 ± 0.6 nM, and 6.9 ± 0.9 nM, respectively. The [ 14 C]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 [ 14 C]taurine uptake in JAR cells. Taurine transport was of high affinity with a K m of 10.2 ± 1.7 μM and fully inhibited by ALX-5407 (IC 50 =522  ± 83 nM). The developed assay is homogenous, rapid, versatile and amenable to automation for the discovery of new neurotransmitter transporter inhibitors.