[3H]-Glycine transport assays

For assaying GlyT2 transport activity, cells were incubated in a solution with an isotopic dilution containing 2 μCi/ml [³H] glycine (1.6 TBq/mmol; PerkinElmer Life Sciences) in PBS, yielding a 10 μM final glycine concentration. To measure unspecific glycine accumulation (background), the transport assay was also performed in the presence of the GlyT2 antagonist ALX1393 (0.4 μM, IC50 = 50 nM). Transport was measured by subtracting the background glycine accumulation in COS7 cells or primary neurons and normalizing to the protein concentration and time of the reaction. Experiments in neurons were performed in the presence of the GlyT1 antagonist NFPS (10 μM) as these cultures present GlyT1 endogenous activity that would contaminate the measurements of GlyT2 transport activity. The reactions were terminated after 10 min by aspiration, followed by washing with HBS.