Glutamate Transport Assay in HeLa Cells

Glutamate transport assay was performed according to our previous study [34 (link), 35 (link)]. HeLa cells in 24 plates transfected with different mutants were washed with 1 mL of choline chloride (ChCl) solution (150 mM ChCl, 5 mM KPi, pH 7.4, 0.5 mM MgSO₄, and 0.3 mM CaCl₂) for two times. Then, NaCl solution (150 mM NaCl, 5 mM KPi, pH 7.4, 0.5 mM MgSO₄, and 0.3 mM CaCl₂) containing 0.4 mCi (0.15 mM) D-[³H]-aspartate (PerkinElmer, Waltham, MA, USA) was added, and the solution was incubated for 10 min at room temperature. Next, 1% SDS was added after washing the cells twice with NaCl solution, and the D-[³H]-aspartate radioactivity was detected. Because both glutamate and aspartate are the substrates for the high-affinity glutamate transport system [77 (link), 78 (link)], the ³H-labeled aspartate could reflect the transport function for glutamate.