Regulating the Conducting States of a Mammalian Serotonin Transporter Michael W Quick  Neuron  Volume 40, Issue 3, Pages 537-549 (October 2003) DOI: 10.1016/S0896-6273(03)00605-6

Figure 1 Syntaxin 1A Regulates SERT Expression and Function (A) Syntaxin 1A eliminates 5HT-induced currents. Representative traces from oocytes expressing SERT alone (top traces) or with syntaxin 1A (bottom traces). Holding potential was −80 mV. Open bars above the traces represent application of 10 μM 5HT. Recordings were made before (left traces) and 15 min after (right traces) injection of 10 ng BoTx. (B) Current-voltage relationship for 5HT-induced currents. Data are plotted from experiments as in (A) with 12–17 oocytes/condition/data point. Error bars are within the symbol size. (C) SERT surface expression. Oocytes expressing both SERT and syntaxin 1A were left untreated or BoTx injected and subjected to surface biotinylation. Immunoblotting was performed on avidin-bound (B) and nonbound (NB) fractions. Data from four such experiments (20 oocytes/condition/experiment) are quantified in the graph relative to total SERT expression. Relative immunoreactivity in BoTx-treated oocytes is significantly different from untreated controls (p < 0.05). (D) Binding of a SERT antagonist to intact oocytes. Oocytes expressing both SERT and syntaxin 1A were left untreated (open symbols) or BoTx injected (filled symbols) and subjected to [125I]RTI-55 binding at various concentrations. Nonspecific binding (squares) was performed in the presence of 50 μM fluoxetine. Specific binding (circles) was calculated by subtracting total binding from nonspecific binding. Data are from six to nine oocytes/condition/data point. Binding parameters based upon Scatchard analysis: Untreated, Kd = 0.61 ± 0.14 nM, Bmax = 20.1 ± 2.4 fmol/oocyte; BoTx-treated, Kd = 0.70 ± 0.22 nM, Bmax = 14.9 ± 2.3 fmol/oocyte. Bmax values were significantly different between groups (p < 0.05). (E) Saturation analysis of 5HT uptake. Oocytes expressing both SERT and syntaxin 1A were left untreated (open symbols) or BoTx injected (filled symbols) and subjected to 5HT uptake at various concentrations. Data are from six to nine oocytes/condition/data point. Uptake parameters based upon Eadie-Hofstee analysis: Untreated, Km = 0.78 ± 0.22 μM, Vmax = 20.3 ± 3.1 fmol/oocyte/sec; BoTx-treated, Km = 0.70 ± 0.22 μM, Vmax = 15.8 ± 2.9 fmol/oocyte/sec. Vmax values were significantly different between groups (p < 0.05). (F) SERT turnover rates. Transport rates for untreated and BoTx-treated oocytes were calculated by dividing Vmax values obtained in (E) by the Bmax values obtained in (D). Neuron 2003 40, 537-549DOI: (10.1016/S0896-6273(03)00605-6)

Figure 2 Syntaxin 1A Changes the Stoichiometry of SERT Transport (A) Charge and substrate flux in SERT-expressing oocytes. Moles of ion flux (charge) and moles of 5HT (10 μM) flux were determined simultaneously in individual oocytes expressing SERT alone. Holding potentials were −40 mV (filled bars) and −80 mV (open bars). Data are from 12 to 17 oocytes/condition. (B) Charge:5HT ratio in oocytes expressing SERT. Data were assessed as in (A). Slope of best-fit line (charges per 5HT molecule) at −80 mV, 12.9; at −40 mV, 7.1. (C) Charge:5HT ratio in oocytes expressing SERT and syntaxin 1A. Data were assessed as in (A) before (open circles) and 20 min after injection of 10 ng BoTx (filled circles). Holding potential was −80 mV. Slope of best-fit line (charges per 5HT molecule): untreated oocytes, 0.02; BoTx, 13.6. (D) Saturation analysis of 5HT uptake for Na+ and Cl−. Oocytes expressing both SERT and syntaxin 1A were left untreated (open symbols) or BoTx injected (filled symbols) and subjected to 5HT uptake at various Na+ (circles) and Cl− (squares) concentrations. Data are from six to seven oocytes/condition/data point. Uptake parameters based upon Eadie-Hofstee analysis: untreated Na+, Km = 25 ± 5 mM; BoTx Na+, Km = 22 ± 6 mM; untreated Cl−, Km = 11 ± 4 mM; BoTx Cl−, Km = 13 ± 3 mM. (E) Charge:5HT ratio at various Na+ and Cl− concentrations. Oocytes expressing both SERT and syntaxin 1A were BoTx injected and assessed as in (A). Holding potential was −40 mV. Data are from five to seven oocytes/condition/experiment. (F) Stoichiometry of flux. Oocytes expressing both SERT and syntaxin 1A were left untreated or BoTx injected, and subjected to uptake using radiolabeled Na+ (22 mM; filled bars), Cl− (22 mM; open bars), or 5HT (10 μM; hatched bars). Holding potential was −40 mV. Data represent SERT-specific uptake (differ ence between total uptake and uptake in sister oocytes treated with 50 μM fluoxetine) from 11 to 19 oocytes/condition from one oocyte batch. Neuron 2003 40, 537-549DOI: (10.1016/S0896-6273(03)00605-6)

Figure 3 Syntaxin 1A Eliminates the Li+-Sensitive Leak but Not Other SERT Currents (A) Representative traces from an oocyte expressing SERT and syntaxin 1A obtained both before (top trace) and 15 min after BoTx injection (bottom trace). Holding potential was −80 mV. Currents were measured in 96 mM Na+ (white bar), 96 mM choline (gray bar), or 96 mM Li+ (black bar) in the absence or presence of 50 μM fluoxetine (hatched bars). (B) Current-voltage relationship for Li+-sensitive leak currents. Data are from experiments as in (A) and plotted as the fluoxetine-sensitive current in 96 mM Na+. Data are from 13 oocytes/condition/data point. (C) The transient current. Representative current-voltage relationship for an oocyte expressing SERT and syntaxin 1A obtained both before (open circles) and 15 min after injection of BoTx (filled circles). The oocyte was voltage clamped at −40 mV, stepped to +60 mV, and then jumped to various potentials from −160 mV to +40 mV. Inset shows kinetics of the decay at −160 mV for an oocyte before and after BoTx treatment. The transient current is eliminated in the presence of fluoxetine (Flu). (D) Current-voltage relationship for H+-sensitive leak currents. Oocytes expressing SERT and syntaxin 1A were measured both before (open circles) and 15 min after injection of BoTx (filled circles). Data are from experiments performed at pH 5.5 and plotted as the fluoxetine-sensitive current in 96 mM choline. Data are from seven oocytes/condition/data point. Neuron 2003 40, 537-549DOI: (10.1016/S0896-6273(03)00605-6)

Figure 4 Syntaxin 1A Binds the N-Terminal Tail of SERT (A) Pull-down assays reveal a physical interaction between SERT and syntaxin 1A. Thalamocortical cell lysates were passed over various GST-syntaxin constructs immobilized on glutathione-agarose beads. Bound proteins were eluted and subjected to immunoblotting using a SERT-specific antibody. (B) Saturation analysis of SERT binding to various syntaxin 1A GST fusion proteins. Experiments were performed as in (A). Percent of SERT bound relative to its binding to 1.0 μM GST-Syn1A was determined from three separate experiments. (C) Pull-down assays reveal a physical interaction between syntaxin 1A and the N-terminal tail of SERT. Thalamocortical cell lysates were passed over various GST-SERT constructs immobilized on glutathione-agarose beads. Bound proteins were eluted and subjected to immunoblotting using a syntaxin 1A-specific antibody. (D) Syntaxin 1A directly binds the N-terminal cytoplasmic tail of SERT. Recombinant syntaxin 1A cytosolic domain (0.4 nmole) was bound to increasing concentrations of GST-N-Tail (open circles) or GST-C-Tail (filled circles) and immunoblotted using a syntaxin 1A-specific antibody. Percent of syntaxin 1A bound relative to its binding to 1.0 μM GST-N-Tail was determined from three separate experiments. (E) N-terminal tail SERT mutants that bind syntaxin 1A. HEK-293 cells transfected with different SERT constructs were passed over GST-Syn1A immobilized on glutathione-agarose beads. Bound proteins were eluted and subjected to immunoblotting using a SERT-specific antibody (upper panel). Surface biotinylation of SERT constructs (lower panel). Data are representative of two separate experiments. (F) Saturation analysis for binding of SERT mutants to GST-Syn1A. Pull-down assays were performed as in (E). Data are plotted relative to binding of wild-type SERT to 1.0 μM GST-Syn1A. Data are from three separate experiments. Neuron 2003 40, 537-549DOI: (10.1016/S0896-6273(03)00605-6)

Figure 5 Stoichiometry of SERT Mutants (A) Current-voltage relationship for 5HT-induced currents. Experiments were performed as in Figure 1A for oocytes expressing syntaxin 1A with either SERT-Δ10 (squares) or SERT-5A (circles). Measurements were made before (open symbols) or 15 min after (filled symbols) BoTx injection. Data are from 9 to 15 oocytes/condition/data point. (B) Turnover rates of SERT mutants. Transport rates were determined for BoTx-treated oocytes expressing syntaxin 1A with either SERT-Δ10 (squares) or SERT-5A (circles). Rates were calculated by dividing Vmax values from Bmax values calculated from uptake and binding saturation analyses, respectively. Data are from two separate experiments, four to six oocytes/data point (as in Figures 1D and 1E). For SERT-Δ10, Vmax = 19.6 ± 4.9 fmol/oocyte/sec, Bmax = 21.4 ± 3.5 fmol/oocyte; for SERT-5A, Vmax = 17.6 ± 3.0 fmol/oocyte/sec, Bmax = 16.7 ± 2.6 fmol/oocyte. (C) Charge:5HT ratio in oocytes expressing SERT mutants and syntaxin 1A. Data are from individual oocytes assessed as in Figure 2A. Holding potential was −40 mV. Symbols are same as in (A). Slope of best-fit lines (charges per 5HT molecule): SERT-Δ10, untreated = −0.04; SERT-Δ10, BoTx = 6.8; SERT-5A, untreated = 7.2; SERT-5A, BoTx = 7.1. (D) Stoichiometry of flux. Oocytes expressing both SERT-5A and syntaxin 1A were left untreated or BoTx injected and subjected to uptake using radiolabeled Na+ (22 mM; filled bars), Cl− (22 mM; open bars), or 5HT (10 μM; hatched bars). Data represent SERT-specific uptake from 8 to 16 oocytes/condition from one oocyte batch. (E) Current-voltage relationship for Li+-sensitive leak currents. Experiments were performed as in Figure 3B and plotted as the fluoxetine-sensitive current in 96 mM Na+. Symbols are same as in (A). Data are from seven oocytes/condition/data point. Neuron 2003 40, 537-549DOI: (10.1016/S0896-6273(03)00605-6)

Figure 6 An N-Terminal Peptide Regulates SERT-Syntaxin 1A Interactions (A) Current-voltage relationship for 5HT-induced currents. Experiments were performed as in Figure 1A for oocytes expressing SERT alone (squares) or with syntaxin 1A (circles). Measurements were made following injection (approximately 5 μM final concentration) of N-Tail (1–30) peptide (filled symbols) or a scrambled control sequence (open circles). Data are from 13 to 21 oocytes/condition/data point. (B) Charge:5HT ratio in oocytes expressing SERT mutants and syntaxin 1A. Experiments were performed as in Figure 2A. Measurements were made at −40 mV following injection of the N-Tail (Scram) or N-Tail (1–30). Slope of best-fit lines (charges per 5HT molecule): N-Tail (Scram) = 0.02; N-Tail (1–30) = 6.9. (C) Stoichiometry of flux. Oocytes expressing both wild-type SERT and syntaxin 1A were acutely injected with N-Tail (1–30) or N-Tail (Scram) and subjected to uptake using radiolabeled Na+ (22 mM; filled bars), Cl− (22 mM; open bars), or 5HT (10 μM; hatched bars). Data represent SERT-specific uptake from 7 to 13 oocytes/condition from one oocyte batch. (D) Current-voltage relationship for Li+-sensitive leak currents. Oocytes expressing both wild-type SERT and syntaxin 1A were acutely injected with N-Tail (1–30) or N-Tail (Scram). Data are plotted as the fluoxetine-sensitive current in 96 mM Na+. Data are from six oocytes/condition/data point. Neuron 2003 40, 537-549DOI: (10.1016/S0896-6273(03)00605-6)

Figure 7 Syntaxin 1A Inhibition of SERT-Mediated Currents in Neurons (A) Saturation analysis of 5HT uptake in thalamocortical neurons. Dissociated cultures 6 DIV were left untreated (open symbols) or treated with 50 μM fluoxetine (filled symbols) and subjected to 5HT uptake at different concentrations. Data are from six wells/data point. Uptake parameters for untreated cultures based upon Eadie-Hofstee analysis: Km = 0.62 ± 0.19 μM, Vmax = 0.022 ± 0.0051 fmol/cell/sec. (B) 5HT-induced currents mediated by SERT. Representative traces of whole-cell patch clamp recordings produced by a voltage step from −60 mV to −120 mV. (Upper left) 10 μM 5HT followed by 10 μM 5HT + 50 μM fluoxetine; (upper right) 10 μM 5HT followed by 3 μM MDMA; (lower left) 10 μM 5HT in the presence, then absence of Na+. (C) SERT-mediated leak currents. Representative traces produced by a voltage step from −60 mV to −120 mV. (Upper) 50 μM fluoxetine; (lower) 50 μM fluoxetine in the absence of Na+. (D) Membrane depolarization by SERT. Neurons were recorded in current-clamp configuration in the presence of 10 μM 5HT followed by 50 μM fluoxetine. Lines connect responses of the same cell. Membrane potential: control, 61.6 ± 1.5; fluoxetine, 65.4 ± 1.2 (p < 0.05). (E) Regulation of SERT currents. Representative traces of whole-cell patch clamp recordings produced by a voltage step from −60 mV to −120 mV measured 1 min (top traces) or 15 min (bottom traces) after gaining cell access. (Left) 10 μM 5HT, 3 μM MDMA, or 10 μM 5HT + 50 μM fluoxetine with pipette solution containing 1 μM N-Tail (1–30); (center) 10 μM 5HT with pipette solution containing 1 μM N-Tail (Scram); (right) 10 μM 5HT with pipette solution containing 350 nM Munc18a. Neuron 2003 40, 537-549DOI: (10.1016/S0896-6273(03)00605-6)