Kv2.1 expression decreases DAT-mediated inward current without reducing surface DAT levels.
A, representative recordings obtained in whole-cell patch-clamp configuration with a voltage step from −120 to −40 mV showing the DAT-mediated inward current in YFP–DAT only cells (top) or cells co-expressing YFP–DAT and mCer3–Kv2.1 (bottom). The DAT-mediated inward current is defined as basal whole-cell current minus the current after nomifensine (10 μm) application, canonically described as nomifensine-sensitive current. B, bar graph shows the DAT-mediated inward current at −120 mV in cells expressing DAT alone (black bar) or in the cells co-expressing DAT plus Kv2.1 (teal bar) (p = 0.0405, n = 8–10; unpaired t test). C, representative immunoblot showing surface DAT (biotinylated fraction) and total DAT (total fraction) from cells with or without Kv2.1 co-expression. D, comparison of surface DAT levels in cells expressing DAT only or cell co-expressing Kv2.1 and DAT. The surface DAT level was determined by calculating the ratio of DAT signal from the biotinylated fraction to total DAT levels. Co-expression of Kv2.1 did not alter the surface DAT level compared with cells without Kv2.1 (p = 0.5496, n = 3, unpaired t test).