The SERT C terminus associates with PDZ proteins and subunits of the COPII coat complex. (A) Protein extracts from mouse brain (10 mg protein) were incubated with 10 μg of either a synthetic peptide that incorporated the 15 C-terminal residues of mouse SERT (SERT-NAV Ct) or a peptide in which the C-terminal valine was replaced by a glutamate (SERT-NAE Ct) and immobilized on Sepharose beads or with Sepharose beads only. Areas of interest of 2D gels representative of three experiments performed independently are illustrated. The arrows indicate the position of spots that were specifically recruited by SERT C terminus. (B) Brain extracts (1 mg protein) were incubated with the indicated Sepharose-immobilized peptides (10 μg each), and proteins recruited by affinity chromatography were analyzed by Western blotting with specific antibodies. The data are representative of three independent experiments.

Cerebral 5-HT uptake is increased in nNOS-deficient and WT mice treated with a peptide preventing the association of SERT with nNOS. (A) [³H]5-HT uptake in synaptosomes from nNOS^Δ/Δ compared with WT mice. Nonspecific uptake was determined in the presence of 1 μM of citalopram. Data represent the means ± SEMs of values obtained in synaptosomes from four WT and four nNOS^Δ/Δ mice. (B) SERT expression in whole-brain and plasma membrane-enriched fractions from WT and nNOS^Δ/Δ mice. Densitometric analysis indicated a 42 ± 3% (n = 3) decrease of SERT in the plasma membrane fraction from WT mice, compared with nNOS^Δ/Δ mice, and no change in total SERT expression. (C) Visualization of intraneuronal accumulation of biotinylated TAT-SERT peptide. (a and b) Avidin and SERT labeling in the raphe nucleus of animals injected with biotinylated TAT-SERT, respectively. (c) Avidin staining in animals treated with saline. (d) Control immunostaining in biotinylated TAT-SERT-injected animals omitting primary (anti-SERT) antibody. (Scale bar: 50 μm.) (D) 5-HT uptake in synaptosomes from mice, which had received two i.v. injections of the TAT-SERT peptide or the TAT-SERTNAE control peptide (500 μg each, separated by a 90-min interval) or saline. The data illustrated are representative of four experiments each performed on different animals. (E) [³H]5-HT uptake in cultured mesencephalic neurons in the absence (control) or presence of either TAT-SERT or TAT-SERTNAE (10 μM, 3 h). Nonspecific uptake was determined in the presence of 50 nM fluoxetine. Data represent the means ± SEMs of values obtained in three experiments. ∗, P < 0.05 vs. control (ANOVA, followed by Dunnett's test). The images illustrate intracellular accumulation of biotinylated TAT-SERT peptide in neurons (Left Upper) and a merge image (Right Upper) of neurons immunolabeled with SERT (red channel) and nNOS antibodies (green channel). (Scale bar: 50 μm.)

nNOS coexpression inhibits 5-HT uptake and decreases plasma membrane SERT density in HEK293 cells. (A) Effect of nNOS coexpression with SERT on [³H]5-HT uptake. Nonspecific uptake was determined in the presence of fluoxetine (10 μM, data not shown). (B) Lack of influence of NO production on the inhibition of 5-HT transport in cells coexpressing nNOS. Cells were treated for 10 min in the absence or presence of A23187 (10 μM) and/or L-NAME (100 μM) before commencing the [³H]5-HT transport assay in the presence of 20 μM of 5-HT. Data represent the means ± SEMs of values obtained in three different experiments performed in quadruplicate (also applies for C and D). ∗, P < 0.05 vs. cells expressing SERT alone (ANOVA, followed by Student-Newman-Keul's test). (C) Production of NO in cells coexpressing SERT and nNOS. Cells were exposed to the treatments indicated in B in the presence of 1 mM 3-isobutyl-1-methylxanthine, and then intracellular cGMP was determined as an index of NO synthesis. ∗, P < 0.0001 vs. basal; †, P < 0.0001 vs. A23187-treated cells. (D) Lack of inhibitory effect on 5-HT transport of nNOS coexpression in cells transfected with a SERT mutant incapable of interacting with nNOS (SERT-NAE). ∗, P < 0.05 vs. cells expressing SERT alone. (E) Effect of nNOS coexpression on cell surface expression of SERT. Biotinylated SERT was detected by Western blotting by using a monoclonal anti-GFP antibody. Intensities of bands in immunoblots were measured by densitometry. Data are the means ± SEMs of three determinations performed on different cultures. P < 0.05 vs. cells expressing SERT alone. (F) Lack of effect of nNOS expression on SERT internalization. Cells biotinylated with sulfo-NHS-SS-biotin were incubated for a 90-min period at 37°C to allow biotinylated SERT internalization, and the remaining cell surface biotin was quenched by using the nonpermeant sulfydryl reagent MESNA. Internalized SERT was detected as described in E. ∗, P < 0.05 vs. cells expressing SERT alone.

5-HT uptake by SERT enhances the activity of coexpressed nNOS in HEK293 cells. (A) HEK293 cells transfected with nNOS alone or cotransfected with SERT and nNOS were treated for 10 min with the indicated concentrations of 5-HT plus 1 mM 3-isobutyl-1-methylxanthine. Note that cGMP was not detectable in cells not transfected with nNOS (data not shown). (B) Cells were preincubated for 10 min in the absence or presence of the 5-HT reuptake inhibitors citalopram (Cital, 3 μM) or paroxetine (Parox, 10 μM) before 5-HT (20 μM) or A23187 (10 μM) exposure. (C) Cells cotransfected with SERT and nNOS were treated for 10 min in the absence or presence of A23187 (10 μM), 5-HT (20 μM), BAPTA-AM (10 μM), W7 (10–50 μM), and calmidazolium (CMZ; 5–25 μM). In experiments using BAPTA-AM, W7, or CMZ, they were added to cells 10 min before A23187 (10 μM) or 5-HT (20 μM). In A–C, data expressed in percentage of basal cGMP level measured in nNOS/SERT cotransfected cells in the absence of any treatment (96 ± 15 fmol/10⁶ cells) represent the means ± SEMs of values obtained in three experiments performed in triplicate on different sets of cultured cells. ∗, P < 0.01, compared with cGMP level in the corresponding condition in the absence of A23187 or 5-HT (ANOVA, followed by Student-Newman-Keul's test). †, P < 0.01, compared with the corresponding value measured in the absence of BAPTA-AM, W7, or CMZ.