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. 2002 Oct 1;22(19):8370-8.
doi: 10.1523/JNEUROSCI.22-19-08370.2002.

Accessibility and conformational coupling in serotonin transporter predicted internal domains

Andreas Androutsellis-Theotokis  1 , Gary Rudnick
Affiliations

Accessibility and conformational coupling in serotonin transporter predicted internal domains

Andreas Androutsellis-Theotokis et al. J Neurosci. .

Abstract

The intracellular topology of serotonin transporter (SERT) was examined using mutants containing single cysteine residues in the predicted cytoplasmic domain of the protein. Cysteine residues in each predicted cytoplasmic domain, including the NH2 and COOH termini and the five predicted internal loops, reacted with methanethiosulfonate (MTS) reagents only when the plasma membrane was permeabilized with digitonin or in membrane preparations but not in intact cells. The reaction was monitored by inactivation of high-affinity binding activity and by incorporation of biotin groups into the protein. Of the seven endogenous cysteine residues predicted to lie in the cytoplasmic domain, modification of only Cys-357 in the third internal loop (IL3) led to loss of activity. Cys-15 in the NH2 terminus and Cys-622 in the COOH terminus also reacted with MTS reagents. Modification of cysteine residues inserted at positions 137 in IL1, 277 in IL2, and 441 in IL4 also led to inactivation, and at positions 157 in IL1 and 532 in IL5, cysteine was modified without an effect on binding activity. These results are in agreement with the originally proposed topology for SERT and argue against an alternative topology proposed for the closely related GABA and glycine transporters. The reactivity of many of the cytoplasmic cysteine residues studied was influenced by ion and ligand binding, suggesting that the internal domains of SERT participate in conformational changes during neurotransmitter transport.

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Figures

Fig. 1.
Fig. 1.
Diagram of predicted topology for SERT. Each amino acid residue is indicated by a filled circle or asquare. The squares show the locations of endogenous cysteine residues. Small arrows point to locations previously determined to react with extracellular cysteine or lysine reagents (Chen et al., 1998). The numbered arrowsshow the residues replaced in X8C or those replaced with cysteine in this work. Striped arrows indicate positions where cysteine replacement led to an inactive transporter. Open arrows indicate positions where cysteine replacement mutants were active but not modified by MTS reagents. Shaded arrows indicate positions where a cysteine was reactive but did not lead to inactivation. Black arrows indicate positions where cysteine was reactive and modification led to partial or complete inactivation.
Fig. 2.
Fig. 2.
Effect of MTS reagents on β-CIT binding and 5-HT transport activities. HeLa cells expressing SERT mutants and membranes prepared from the same cells were treated with the indicated concentrations of MTSEA or MTSET for 15 min in PBS/CM, washed, and assayed for the remaining binding and transport activities. Transport and binding activities are shown as a percentage of the uninhibited level for the same mutant as shown in Table 1.
Fig. 3.
Fig. 3.
Effect of plasma membrane permeabilization on inactivation by MTSET. Intact HeLa cells expressing SERT mutants were treated, where indicated, for 4 min with 0.0025% digitonin in PBS/CM, washed once with PBS/CM, and then incubated with 5 mm MTSET for 10 min. After incubation, the cells were washed three times with PBS/CM and collected by scraping, and crude membrane fractions were prepared as described in Materials and Methods for binding activity determinations. Data are means ± SDs from six measurements in three separate experiments expressed as the percentage of transport or binding activity relative to the control samples without digitonin or MTSET. The asterisks indicate significant differences in inactivation as a result of digitonin addition (p < 0.05 in the paired Student'st test).
Fig. 4.
Fig. 4.
Protection by serotonin and cocaine from MTSEA inactivation of β-CIT binding. Membranes from HeLa cells expressing SERT mutants (X8C-L137C, X8C-S277C, X8C-I357C, and X8C-A441C) were assayed for binding activity after a 15 min incubation with MTSEA (1, 0.01, and 0.005 mm MTSEA for X8C-L137C, X8C-S277C, X8C-I357C, and X8C-A441C, respectively, when the inactivation was performed at room temperature, and 5, 0.05, 5, and 0.025 mm, respectively, when the inactivation was performed at 4°C). Before addition of MTSEA, 10 mm of either serotonin or cocaine was added for X8C-L137C, X8C-S277C, and X8C-A441C, and 4 mm of serotonin or 7 mm of cocaine was added for X8C-I357C. After the MTSEA incubation, membranes were washed five times and assayed for β-CIT binding activity. The bar graph shows residual binding activity. Protection is evidenced by the increased residual activity after MTSEA treatment. Also shown is residual activity in the presence and absence of ligands when the inactivation was performed at low temperature (Na+, 4o) or in the absence of Na+(NMDG+). The left three sets ofcolumns represent data from X8C-L137C, the next three from X8C-S277C, the next three from X8C-I357C, and the right three from X8C-A441C. Data are means ± SDs from six measurements in three separate experiments. The asterisks indicate significant protection by 5-HT or cocaine (p < 0.05 in the paired Student'st test).
Fig. 5.
Fig. 5.
Effect of monovalent cations on the inactivation by MTSEA. Membrane preparations from HeLa cells expressing SERT mutants (X8C-L137C, X8C-I157C, X8C-S277C, X8C-357C, and X8C-A441C) were treated for 15 min with MTSEA (1.5, 2, 0.01, 1, and 0.004 mm, respectively) in binding buffer (Na+) and in buffer in which all Na+ was replaced by NMDG+, K+, Li+, Cs+, or Rb+. After incubation, the membranes were washed three times with Na-containing binding buffer, and β-CIT binding was subsequently measured. The bar graph shows residual binding activity. Data are means ± SDs from four measurements in two separate experiments. The asterisks indicate significant increase in rate relative to NMDG (p < 0.05 in the paired Student'st test).
Fig. 6.
Fig. 6.
Visualization of immunoprecipitated C109A SERT mutant with biotinylated anti-Flag antibodies and with HRP-labeled streptavidin. A, HeLa cells transiently transfected with SERT C109A cDNA or mock-transfected (no DNA) were permeabilized by addition of 0.0025% digitonin for 4 min at 25°C. Some samples (marked Biotin) were treated with MTSEA-biotin. All samples were washed three times with PBS/CM and immunoprecipitated with antibodies against the N-terminal myc tag of C109A. Samples were resolved by nonreducing SDS-PAGE and transferred to a nitrocellulose membrane. Lanes marked FLAG were treated with biotin-linked antibodies against the C-terminal FLAG tag of C109A, and then all samples were visualized with HRP-labeled streptavidin. Themarks indicate the mobility of standards of the following molecular sizes: 184, 84, 62, and 38 kDa. B, HeLa cells transiently transfected with C109A cDNA were treated as follows: lanes 1 and 2, cells were treated with MTSEA-biotin (0.5 mm, 10 min) with or without previous permeabilization using digitonin (0.0025%, 4 min), washed three times with PBS/CM, and then solubilized and immunoprecipitated with anti-myc; lanes 3 and 4, cells were treated the same as lanes 1 and 2, but before solubilization they were first homogenized to prepare membranes.Lane 5, Cells were homogenized without previous permeabilization of the plasma membrane, and then this crude membrane fraction was treated with MTSEA-biotin (∗) and washed three times with PBS/CM, and SERT protein was solubilized and immunoprecipitated.C, Membrane preparations from cells expressing SERT mutants X8C and C109A were treated with 1 mm MTSEA-biotin for 10 min at room temperature, washed three times with PBS/CM by centrifugation, solubilized, and immunoprecipitated with anti-myc, and biotin label was subsequently detected by HRP-labeled streptavidin as described above. D, Cell surface biotinylation of X8C-transfected cells. Cells expressing X8C were treated with sulfo-NHS-LC-biotin as described previously (Kilic and Rudnick, 2000). Samples were washed three times with PBS/CM, solubilized, and immunoprecipitated with anti-myc, and biotin label was subsequently detected by HRP-labeled streptavidin as described above.
Fig. 7.
Fig. 7.
Biotinylation of single cysteines. Cells were treated with (+, or no label) or without (−) digitonin (0.0025%) for 4 min, treated with MTSEA-biotin or MTSEA-biotinCap, as indicated, solubilized, immunoprecipitated, and visualized as described. The resulting signal is shown as an integrated density. In the absence of digitonin, no signal was observed with any mutant, and for simplicity, only the lane corresponding to C109A is shown. The mutants X8C-S277C and X8C-357C were labeled much more efficiently by MTSEA-biotinCap. Mutant names are abbreviated so that X8C-L137C is shown asX137C, etc. Underlining indicates cysteine residues not present in the native SERT sequence. Results are averages from two experiments.
Fig. 8.
Fig. 8.
Ion dependence of biotinylation at cytoplasmic cysteine residues. Membrane preparations from cells expressing the indicated mutants were biotinylated either in Na+-containing buffer or in buffer in which Na+ was replaced by K+. The resulting signal is shown as an integrated density.Underlining indicates cysteine residues not present in the native SERT sequence. Results are averages from two experiments.
Fig. 9.
Fig. 9.
Reactivation of binding activities and removal of biotin signal of SERT mutants by 12 mm free cysteine after inactivation by MTS reagents. Membranes from HeLa cells expressing SERT mutants (X8C-L137C, X8C-S277C, X8C-357C, and X8C-A441C) were treated with MTSEA-biotinCap at the indicated concentrations for 15 min. Subsequently, the membranes were washed once, 12 mm free cysteine was added to the membranes for different time durations (0, 5, 10, 15, 25, 40, and 60 min), and then the membranes were assayed for β-CIT binding activity. Data are means ± SDs from six measurements in three separate experiments expressed as the percentage of binding activity relative to the uninhibited controls att = 20 min (maximal reactivation). Additionally, membranes from HeLa cells expressing SERT mutants (X8C-L137C, X8C-I157C, X8C-S277C, X8C-357C, and X8C-A441C) were treated with 1.5 mm MTSEA-biotinCap for 10 min, washed, and then treated with or without 12 mm free cysteine for 20 min. SERT protein was resolved by SDS-PAGE as described in Materials and Methods and visualized by streptavidin-HRP. The asterisk indicates a significant increase in activity after cysteine treatment (p< 0.05; paired student's t test).
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