Sucic S , Dallinger S , Zdrazil B , Weissensteiner R , Jørgensen TN , Holy M , Kudlacek O , Seidel S , Cha JH , Gether U , Newman AH , Ecker GF , Freissmuth M , Sitte HH .

P01 DA 12408 NIDA NIH HHS , F 3506 Austrian Science Research Fund, F 3510 Austrian Science Research Fund, F 3506 Austrian Science Fund FWF, F 3510 Austrian Science Fund FWF, F 3502 Austrian Science Fund FWF, P01 DA012408 NIDA NIH HHS

	FIGURE 1. Expression and function of wild type SERT and of mutated versions that lack candidate N-terminal phosphorylation sites. A, representative confocal laser scanning microscopy images illustrating that the cellular expression of SERTS62A and SERTT81A is comparable with that of wild type SERT. All constructs are almost exclusively targeted to the plasma membrane (also see for the ratio staining of plasma membrane to cell interior in Table 2). HEK293 cells were transiently transfected with CFP-tagged transporter constructs; the cell membrane was visualized by trypan blue staining (26). B, saturation of [3H]5HT uptake by SERT and the mutants expressed in transiently transfected CAD cells. Nonspecific uptake was determined in the presence of 10 μm paroxetine (n = 3–6, each experiment performed in triplicate).
	FIGURE 2. Amphetamine-stimulated release of [3H]5HT by wild type SERT (A), SERTS62A (B), SERTT81A (C), SERTR79A (D), and SERTT81D (E). Transiently transfected CAD cells expressing wild type and mutant SERTs were preloaded with [3H]5HT for 30 min at 37 °C and subsequently superfused (as described under “Experimental Procedures”). Upon reaching a stable base line, three 2-min fractions (6 min) were collected to define basal efflux (basal efflux, mean of the three fractions before PCA addition: wild type SERT, 0.42 ± 0.01%·min−1, i.e. 296 ± 10 dpm·min−1, n = 99 observations of randomly chosen experiments performed on different days; SERTS62A; 0.65 ± 0.04%·min−1, i.e. 480 ± 38 dpm·min−1, n = 67; SERTT81A, 1.77 ± 0.14%· min−1, i.e. 517 ± 22 dpm·min−1, n = 99; SERTR79A, 1.28 ± 0.05%·min−1, i.e. 563 ± 29 dpm·min−1, n = 41; and SERTT81D, 1.06 ± 0.04%·min−1, i.e. 318 ± 21 dpm·min−1, n = 104). Thereafter, cells were exposed to PCA (10 μm).
	FIGURE 3. Concentration-response curve for amphetamine-induced release in wild type monoamine transporters and juxtamembrane threonine mutants. A, PCA-stimulated [3H]5HT release in wild type and mutant SERT. B and C, d-amphetamine (D-AMPH)-stimulated release of [3H]MPP+ by wild type DAT, DATT62A, and DATT62D (B) and wild type NET, NETT58A, and NETT58D (C). The experiments were performed as described in the legend to Fig. 2 and under “Experimental Procedures” (basal efflux for wild type DAT, 0.25 ± 0.01%·min−1, i.e. 255 ± 8 dpm·min−1, n = 148; DATT62A, 0.29 ± 0.02%·min−1, i.e. 226 ± 4 dpm·min−1, n = 132; DATT62D, 0.17 ± 0.01%·min−1, i.e. 105 ± 4 dpm·min−1, n = 144. Basal efflux for wild type NET, 0.039 ± 0.002%·min−1, i.e. 47 ± 2 dpm·min−1, n = 48; NETT58A, 0.074 ± 0.002%·min−1, i.e. 73 ± 2 dpm·min−1, n = 47; NETT58D, 0.065 ± 0.009%· min−1, i.e. 69 ± 15 dpm·min−1, n = 39).
	FIGURE 4. FRET in wild type SERT and SERT-T81A. Transiently transfected CAD cells co-expressing wild type C-SERT and Y-SERT (labeled SERT/SERT), C-SERT and Y-SERTT81A (SERTT81A/SERT), or C-SERTT81A and Y-SERTT81A (SERTT81A/SERTT81A) were grown on glass coverslips. FRET was recorded as outlined under “Experimental Procedures.” The C-SERT-Y construct was used as positive control, whereas CFP co-expressed with YFP represented the negative control in each experiment. The NFRET values from at least three individual experiments (with 5–10 visible fields) were calculated, and there were no statistical differences (p > 0.05, one-way ANOVA followed by Tukey's post hoc t tests) between NFRET values for the following conditions: SERT/SERT, SERTT81A/SERT, and SERTT81A/SERTT81A.
	FIGURE 5. Amphetamine-induced currents (A), membrane depolarization (B), quantification by binding of the fluorescent inhibitor JHC1-64 (C), and [3H]5HT release (D) by the wild type and mutant SERT expressed in X. laevis oocytes. A, current/voltage (I-V) relations were obtained by jumping from a holding potential (Vhold) of −40 mV to −100 mV followed by a ramp to +40 mV over a total time of 10 s (n = 9). The PCA-induced current was defined as the current measured in the presence of PCA (10 μm) after subtraction of the current measured in the corresponding control buffer (IPCA − Icontrol). B, membrane potentials recorded during release experiments under current clamp conditions for wild type SERT and SERTT81A. The error bars represent the standard deviation (p < 0.05; Mann-Whitney U test, two-tailed; n = 3). C, saturation curves for JHC1-64 binding to X. laevis oocytes expressing wild type SERT, SERTT81A, and Δ64SERT. Oocytes (that had been used for voltage clamp experiments) were subsequently incubated in a cumulative manner with increasing concentrations of JHC1-64 for 200 s. Images were captured using the same configuration for all cells, with the indicated concentrations of JHC1-64. D, PCA-induced release of [3H]5HT in cRNA-injected X. laevis oocytes under current clamp conditions (n = 4).
	FIGURE 6. Molecular dynamics simulations of SERTThr-81 mutants reveal models favoring inward facing states. A, snapshot of wild type SERT after 16 ns of MD simulation. The Thr81 side chain forms a stable H-bond with the backbone carbonyl of Tyr350 in IL3. B, snapshot of SERTT81A after 6 ns of MD simulation; the H-bond is not formed between Ala81 and Tyr350 during the course of the simulation. C, snapshot of SERTT81D after 6 ns of MD simulation; no H-bond is formed between Asp81 and Tyr350 during the course of the simulation. D, snapshots of wild type SERT and SERTT81D, following an MD simulation (backbones superimposed). In the in silico mutant (displayed in orange), the cytoplasmic region of TM1 is moved apart from TMD6 and IL3 (indicated by magenta arrows) in comparison with wild type SERT (green). E and F, snapshots of wild type SERT and SERTT81D during MD production simulation (backbones superimposed); TM12 moves away from TM1 (N terminus) after mutation of Thr81 to alanine or aspartate (wild type SERT, green; SERTT81A, yellow; SERTT81D, orange; Cα atoms of Thr81 are displayed in sphere representation).
	FIGURE 7. Probing the conformational state of SERTT81A by measuring the affinity of ibogaine (A) and by using intramolecular FRET as a molecular ruler (B). A, inhibition by ibogaine of [3H]imipramine binding to membranes (10–15 μg) prepared from HEK293 cells expressing wild type SERT or SERTT81A. The incubation was done in a final volume of 0.25 ml containing 20 mm Tris-HCl, pH 7.4, 1 mm MgCl2, 120 mm NaCl, and 5 and 1 nm [3H]imipramine for 20 min at 25 °C. Specific binding in individual experiments amounted to 1500–2000 cpm and was set at 100% to normalize for inter-assay variation; nonspecific binding (determined in the presence of 10 μm paroxetine) was ≤200 cpm. Data are means ± S.D. of three independent experiments carried out in duplicate. B, Thr81 was mutated in a SERT construct tagged with CFP and YFP at the N and C termini, respectively. FRET was recorded microscopically in HEK293 cells transiently expressing C-SERT-Y and C-SERT-YT81A as outlined in the legend to Fig. 4. A significant reduction in the NFRET value (determined with Image J PixFRET software as outlined under “Experimental Procedures”) was observed for the mutant in comparison with the wild type SERT (p = 0.0052, from three individual transfections comprising a total of 22 (wild type) and 26 (mutant SERT) cells, unpaired t test).
	FIGURE 8. Binding of [3H]imipramine to wild type SERT and SERTT81A (A, C, and D) and of [3H]CFT to wild type DAT and DATT62A (B). A and B, saturation binding experiments were carried out in a final volume of 0.25 ml containing membranes expressing wild type (5 μg) and mutant SERT (7 μg) or wild type and mutant DAT (20 μg). The reaction was carried out in 20 mm Tris-HCl, pH 7.4, 1 mm MgCl2, 120 mm NaCl, 3 mm KCl and the indicated concentrations of [3H]imipramine for 10 min at 25 °C. For binding of [3H]CFT, the assay indications were similar except that the buffer did not contain KCl and it was supplemented with 10 μm ZnCl2 as indicated (empty symbols in B). Shown are representative experiments carried out in duplicate that were replicated at least three times. C, reaction was done at a constant protein concentration (40 μg/ml), but the reaction volume was adjusted to 0.15 and 0.5 ml for 10 and 3 nm [3H]imipramine, respectively. At the time point indicated, the reaction was terminated by rapid filtration over glass fiber filters precoated with 1% polyethyleneimine. D, pseudo-first order rate constant (kobs) calculated from experiments carried out as outlined for 3 and 10 nm in C was plotted as a function of ligand concentrations. Shown are data obtained in three independent experiments that were carried out in parallel for wild type and mutant SERT; error bars represent the standard error of the estimate calculated for each individual curve.
	FIGURE 9. Expression of A64SERT and TACSERT in comparison with wild type SERT. The schematic representation illustrates the nature of the constructs employed in our study. The star indicates the position of Thr81. HEK293 cells were transiently transfected with cDNA encoding wild type or the Δ64SERT truncation mutant tagged with CFP (A and B) or the TACSERT (C). The cell surface expression of CFP-tagged transporters (A and B) was visualized by confocal microscopy by sequentially capturing images for CFP (green) and trypan blue (red; to delineate the cell surface). Overlays (in yellow) highlight the cell surface expression of the transporters. C, expression of TACSERT was visualized by staining fixed, nonpermeabilized cells with the M1 antibody directed against the N-terminal FLAG epitope followed by a secondary Alexa488-conjugated goat anti-mouse antibody using a Zeiss LSM510 confocal microscope (left image; the scale bar indicates 10 μm) as outlined under “Experimental Procedures”; the cell surface is shown in a bright field image (right image).
	FIGURE 10. Specific [3H]5HT uptake (A), abolition of amphetamine-stimulated [3H]5HT release (B), and marked reduction in the affinity of [3H]imipramine (C), resulting from the truncation of the SERT N terminus. A, uptake of [3H]5HT was determined in HEK293 cells transiently expressing wild type SERT and Δ64SERT as outlined in the legend to Fig. 1. B, PCA-induced release was determined as in Fig. 2, and the concentration-response curves generated as in Fig. 3. C, saturation of [3H]imipramine binding was performed with membranes (20 μg) prepared from HEK293 cells transiently expressing wild type SERT and Δ64SERT as outlined in the legends to Figs. 7 and 8.
	FIGURE 11. Constraining the flexibility of the amino tail in TACSERT does not impair substrate uptake (A) but does preclude PCA-induced efflux (B). A, uptake of [3H]5HT was determined in HEK293 cells expressing wild type SERT and TACSERT as outlined in the legend to Fig. 1. B, comparison of [3H]5HT efflux induced by PCA in TACSERT and wild type SERT. HEK293 cells transiently expressing the indicated versions of SERT were preloaded with [3H]5HT as described in Fig. 2. Release was induced by superfusing the cells with 10 μm PCA. The results are presented as bar graphs showing specific uptake (A, n = 3 from 3 individual experiments) or base-line-subtracted efflux (B, n = 8 from two independent experiments).

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