title: 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 [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.
authors.affiliation: Department of Neuroscience West Point, Merck & Co. Inc., West Point, PA 19486, USA.
authors.forename: Jacinta B
authors.initials: JB
authors.lastname: Williams
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Mallorga
authors.forename: Wei
authors.initials: W
authors.lastname: Lemaire
authors.forename: David L
authors.initials: DL
authors.lastname: Williams
authors.forename: Sang
authors.initials: S
authors.lastname: Na
authors.forename: Smita
authors.initials: S
authors.lastname: Patel
authors.forename: P Jeffrey
authors.initials: PJ
authors.lastname: Conn
authors.forename: Jeffrey P
authors.initials: JP
authors.lastname: Conn
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Pettibone
authors.forename: Christopher
authors.initials: C
authors.lastname: Austin
authors.forename: Cyrille
authors.initials: C
authors.lastname: Sur
mesh_terms: 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
chemical_list: 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