Skip to main content
. 2021 Sep 22;6(18):e151496. doi: 10.1172/jci.insight.151496

Figure 2. DAT-K619N displays functional impairments and reduced surface expression in vitro.

Figure 2

(AC) Evaluation of DAT-K619N functions and surface expression in transiently transfected HEK293 cells. (A) Functional comparison of DAT-K619N to WT using [3H]-dopamine (DA) uptake. Uptake curves (left) are average curves of 6 experiments each performed in triplicate and normalized to the fitted maximal uptake capacity (Vmax) of DAT-WT. The DAT-K619N variant demonstrated reduced Vmax (right bar diagram) compared with DAT-WT (P < 0.01, 1-sample 2-tailed t test, n = 6) with no accompanying change in Km (Km =1.4 ± 0.3 μM for K619N vs. 1.7 ± 0.4 μM for DAT-WT, P > 0.05 Student’s t test). (B) Amphetamine-induced amperometric currents with representative traces of amperometric currents (left) and quantification of the amphetamine-induced peak currents relative to DAT-WT (right). DA-release through DAT-K619N was impaired compared with DAT-WT (P < 0.05, 1-sample 2-tailed t test, n = 9 for WT and n = 7 for DAT-K619N). (C) Surface biotinylation of transiently transfected HEK293 cells. The amount of DAT-K619N relative to DAT-WT was decreased in the surface and the total protein fractions (P < 0.05, 1-sample 2-tailed t test, n = 4). (D) Confocal live imaging of surface-expressed WT and DAT-K619N in HEK239 cells by labeling with the fluorescent cocaine analog, JHC 1-64 (20 nM). Mean intensity of the JCH 1-64 signal was reduced for DAT-K619N relative to DAT-WT. Images were acquired from 4 independent transfections, and intensities were normalized to WT mean intensity for each imaging session (P < 0.05, 1-sample 2-tailed t test, n = 21 images per group). Data are mean ± SEM. *P < 0.05, **P < 0.01.