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

Williams JB, Mallorga PJ, Lemaire W, Williams DL, Na S, Patel S, Conn PJ, Conn JP, Pettibone DJ, Austin C, Sur C
Anal Biochem. 2003 321 (1): 31-7

PMID: 12963052 · DOI:10.1016/s0003-2697(03)00431-7

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+/-3 microM. 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+/-4.6 microM, 2.8+/-0.6 nM, and 6.9+/-0.9 nM, 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+/-1.7 microM 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.

MeSH Terms (16)

Amino Acid Transport Systems, Neutral Carbon Radioisotopes Cell Count Cell Line, Tumor Chlorides Dose-Response Relationship, Drug Female Gene Expression Regulation, Neoplastic Glycine Glycine Plasma Membrane Transport Proteins Humans Pregnancy Scintillation Counting Sodium Taurine Time Factors

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