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 [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.
Williams
Jacinta B
JB
Department of Neuroscience West Point, Merck & Co. Inc., West Point, PA 19486, USA.
Mallorga
Pierre J
PJ
Lemaire
Wei
W
Williams
David L
DL
Na
Sang
S
Patel
Smita
S
Conn
P Jeffrey
PJ
Conn
Jeffrey P
JP
Pettibone
Douglas J
DJ
Austin
Christopher
C
Sur
Cyrille
C
D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002452:Cell Count; D045744:Cell Line, Tumor; D002712:Chlorides / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005998:Glycine / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D050487:Glycine Plasma Membrane Transport Proteins; D006801:Humans; D011247:Pregnancy; D012588:Scintillation Counting / Q000379:methods*; D012964:Sodium / Q000494:pharmacology*; D013654:Taurine / Q000378:metabolism; D013997:Time Factors
D026921:Amino Acid Transport Systems, Neutral; D002250:Carbon Radioisotopes; D002712:Chlorides; D050487:Glycine Plasma Membrane Transport Proteins; C493268:SLC6A9 protein, human; D013654:Taurine; D012964:Sodium; D005998:Glycine