Fig. 9
From: The sigma-1 receptor modulates methamphetamine dysregulation of dopamine neurotransmission
σ1R is co-localized with the dopamine transporter at or near the plasma membrane. a Representative confocal image of immunolabeled endogenous DAT (green) and endogenous σ1R (red) in primary culture of dopaminergic neurons shows DAT and σ1R are co-localized (shown in orange). The Pearson’s coefficient = 0.534 ± 0.03, n = 24 neurons (scale bar: 25 μm). Total internal reflection fluorescence microscopy (TIRFM) imaging of the neuronal cell body shows co-localization occurring at or near (<150 nm) the plasma membrane (right panel). b TIRFM imaging of cells expressing CFP-DAT and σ1R-YFP reveals localization of the two proteins at or near the plasma membrane (scale bar: 25 μm). c Super-resolution STED microscopy of σ1R-YFP expressing cells. The inset shows a 200% magnification of the indicated region revealing a σ1R-YFP hot-spot at the cellular edge (scale: 5 µm). d YFP-DAT cells expressing σ1R-CFP shows areas of the endoplasmic reticulum (as indicated by σ1R-CFP expression) in close contact with the plasma membrane (as indicated by YFP-DAT expression; scale bar 25 µm). The right panel shows a 500% magnification of the indicated region. Arrows highlight the areas of contact between σ1R-CFP and YFP-DAT. e FRET efficiency (FRETeff) analysis reveals an interaction between σ1R-CFP and YFP-DAT. While 1 μM PRE-084 (30 min), 1 μM BD-1063 (30 min), or METH (10 μM, 15 min) alone did not increase the FRETeff compared to control, combined PRE-084 and METH treatment significantly increased the association between σ1R-CFP and YFP-DAT as measured by an increased FRETeff. 1 μM BD1063 pretreatment before PRE-084/METH blocked the effect of PRE-084. (n = 10–19 cells per group; F (5,75) = 2.4, P = 0.0005, one-way ANOVA; Tukey’s test for multiple comparisons; P = 0.1694 comparing PRE-084/METH to BD1063/PRE-084/METH). f Nonspecific FRET was determined in cells expressing YFP-DAT/empty CFP-vector or σ1R-CFP/YFP-vector (n = 5 cells). Additionally, σ1R-YFP co-expressed with CFP-transferrin receptor (CFP-TfR) did not result in detectable FRET (n = 4 cells). For positive control, the CFP-YFP fusion protein FRET8 was used (n = 5 cells). **P < 0.01. Data is represented as mean ± SEM
