In Vitro Assays for the Functional Characterization of the Dopamine Transporter (DAT)

Detailed in this unit are protocols for studying the in vitro uptake of dopamine (DA) as a means for defining the functional characteristics of dopamine transporters. All assays are performed using commercially available cell lines that transiently express the transporter under investigation. The three main assays provided are: a kinetic assay to calculate the affinity (K_M ) and maximal velocity (V_max ) of radiolabeled DA uptake into cells; concentration-response assays to measure the potencies (IC₅₀ /K_i values) of test compounds as transport inhibitors; and an efflux assay to assess the ability and potency (EC₅₀ ) of a ligand to elicit reverse transport of DA accumulated in the cell. Although the methods are described using DAT and its ligands, the same procedure can be employed for studying serotonin and norepinephrine transporters as well. © 2017 by John Wiley & Sons, Inc.