We used a YFP-tagged version of DAT and a Nikon laser scanning confocal microscope system to study DAT-internalization following inhibitor binding. Plasma membrane was stained with 0.4% Trypan blue (Sigma) for 5 min.
Kinetic modeling was performed as done previously (85 (link)). Systems Biology Toolbox and MATLAB 2015a (Mathworks) were used to study the time-dependent non-competitive pharmacology of the inhibitors.
Transporter electrophysiology was used to resolve the dissociation constant of the tested inhibitors in vitro and to assess the dependence of inhibitor binding on transporter state/s as reported previously (57 (link), 86 (link)). Transporter-mediated current was recorded from HEK293 cells expressing DAT, in the whole-cell configuration. Cells were clamped at −60 mV and continuously superfused with the necessary solutions.
Kinetic modeling was performed as done previously (85 (link)). Systems Biology Toolbox and MATLAB 2015a (Mathworks) were used to study the time-dependent non-competitive pharmacology of the inhibitors.
Transporter electrophysiology was used to resolve the dissociation constant of the tested inhibitors in vitro and to assess the dependence of inhibitor binding on transporter state/s as reported previously (57 (link), 86 (link)). Transporter-mediated current was recorded from HEK293 cells expressing DAT, in the whole-cell configuration. Cells were clamped at −60 mV and continuously superfused with the necessary solutions.
