Direct PIP2 binding mediates stable oligomer formation of the serotonin transporter

The human serotonin transporter (hSERT) mediates uptake of serotonin from the synaptic cleft and thereby terminates serotonergic signalling. We have previously found by single-molecule microscopy that SERT forms stable higher-order oligomers of differing stoichiometry at the plasma membrane of living cells. Here, we report that SERT oligomer assembly at the endoplasmic reticulum (ER) membrane follows a dynamic equilibration process, characterized by rapid exchange of subunits between different oligomers, and by a concentration dependence of the degree of oligomerization. After trafficking to the plasma membrane, however, the SERT stoichiometry is fixed. Stabilization of the oligomeric SERT complexes is mediated by the direct binding to phosphoinositide phosphatidylinositol-4,5-biphosphate (PIP2). The observed spatial decoupling of oligomer formation from the site of oligomer operation provides cells with the ability to define protein quaternary structures independent of protein density at the cell surface.

T he human serotonin transporter (SERT) is a 12-pass transmembrane protein targeted to presynaptic nerve terminals and belongs to the neurotransmitter: sodium symporter (NSS) or solute carrier 6 (SLC6) family 1 . These transmembrane proteins mediate the high-affinity uptake of neurotransmitters from the synaptic cleft and are, hence, of pivotal importance for synaptic signal transmission by terminating chemical signal transduction between neurons. SERT is the target for antidepressants like serotonin-selective reuptake inhibitors 2 as well as for illicitly used drugs such as amphetamines 3 ; the latter act by reversing the transport direction of SERT, provoking release of serotonin (5-HT) into the extracellular space 4 .
Biochemical and spectroscopic studies have reported that SERT is present as oligomeric complexes at the plasma membrane [5][6][7][8][9] . Likewise, oligomerization of a number of other NSS family members has been described 10 . The oligomeric state, however, does not seem to be crucial for uptake activity: for example, it was found that oligomerization-deficient mutants of the GABA transporter (GAT1) retain unchanged transport activity 11 . Currently, two possible roles of NSS oligomerization are discussed: (i) oligomerization of correctly folded proteins is necessary to pass the quality control for trafficking from the endoplasmic reticulum (ER) 12 , in the case of SERT specifically by allowing the interaction with SEC24C (refs 13,14). (ii) It has been reported that oligomerization is a prerequisite for the reverse operation of the transporter which affords substrate release 15 .
Using single-molecule fluorescence microscopy, we have previously discovered that SERT forms a broad distribution of assemblies ranging from monomers up to pentamers 7 . The homo-association at the plasma membrane did not depend on SERT surface density and was stable at least over 10 min. We proposed a model based on kinetic trapping of oligomers at the plasma membrane, subsequent to an equilibration which occurred at an unknown subcellular organelle. The site of equilibration and the mechanism behind kinetic trapping, however, remained unclear.
Some arguments pointed our interest to the negatively charged phospholipid phosphatidylinositol-4,5-biphosphate (PIP 2 ). PIP 2 is part of a number of signalling pathways, for example, endo-and exocytosis, cell adhesion, cell motility, phagocytosis or G protein-coupled receptor signalling 16 . It is a minor phospholipid that is mainly found at the cytoplasmic leaflet of the plasma membrane, where it occurs at a surface density of about s PIP B20,000-60,000 molecules mm À 2 (ref. 17). Pronounced differences exist in the subcellular localization of PIP 2 : PIP 2 comprises about 1% of total lipid at the plasma membrane 17 , whereas only trace amounts of PIP 2 are present at the ER 18 . PIP 2 binding to transmembrane proteins is frequently observed, for example, for ion channels, where it regulates channel activity by influencing the open probability 19 . Likewise, we have recently found that the functional activity of SERT and the dopamine transporter (DAT) was influenced by PIP 2 binding 20,21 : upon enzymatic depletion of PIP 2 or mutation of identified PIP 2 binding sites, amphetamine-induced substrate efflux was markedly reduced whereas uptake rates were essentially unaffected. Similarly, while the oligomeric configuration does not seem to influence neurotransmitter uptake 11 , amphetamineinduced neurotransmitter release has been shown to rely on the quaternary arrangement 15 .
In the present study, we quantify the degree of SERT oligomerization at different subcellular localizations of Chinese hamster ovary (CHO) cells and analyse the kinetics of protomer turnover. We find evidence that SERT oligomers are pre-formed at the ER following a dynamic equilibrium model. At the plasma membrane, kinetic trapping arrests the oligomers at the pre-set stoichiometry. Our data suggest that the different subcellular concentrations of PIP 2 mediate the differential SERT oligomerization behaviour, likely by direct physical connection of SERT protomers.

SERT oligomerization depends on subcellular localization.
First, we addressed if SERT oligomerization differs between the plasma membrane and the ER. Monomeric GFP (mGFP) was inserted at the cytosolic N-terminus of SERT to allow for visualization (mGFP-SERT) 22 . Plasma membrane-localized mGFP-SERT was recorded at the bottom cell membrane via total internal reflection fluorescence (TIRF) microscopy. To retain SERT in the ER, we overexpressed a dominant-negative mutant of Sar1a (Sar1a-T39N) 13 , a small GTPase that regulates the assembly of COPII vesicles for plasma membrane trafficking. Sar1a-T39N is a GDP-restricted mutant which prevents the formation of COPII in a dominant-negative manner, thereby arresting SERT in the ER. ER-retained mGFP-SERT was studied at junctions between ER and the plasma membrane, where singlemolecule tracking at high signal-to-noise ratio using TIRF microscopy is feasible 23,24 .
We used the 'Thinning out clusters while conserving stoichiometry' (TOCCSL) technique previously established in our lab 7,[25][26][27] to determine the oligomeric state of mGFP-SERT. TOCCSL extends fluorescence recovery after photobleaching (FRAP) to the level of single-molecule fluorescence microscopy (Fig. 1a). Typically, the high density of fluorescently labelled proteins results in a homogenously labelled surface, thereby precluding direct single-molecule measurements (Fig. 1a,i). In TOCCSL, a small area of the cell membrane is irreversibly photobleached by a strong laser pulse focused through a rectangular pinhole onto the sample (Fig. 1a,ii,iii). The high laser intensity completely abrogates the fluorescence signal of the mGFPs in every SERT molecule within the illuminated area, while retaining full brightness outside this area. During the subsequent recovery phase unbleached molecules and oligomers diffuse into the previously bleached area due to Brownian motion (Fig. 1a,iv). In contrast to FRAP experiments, however, we exploit in TOCCSL the very onset of this recovery process, when individual molecules can be monitored as single, clearly distinguishable fluorescent spots (Fig. 1a,v). The brightness of these spots was determined and compared with the brightness of single mGFP-SERT molecules recorded under the same conditions and in the same subcellular compartments, yielding the statistical distribution of mGFP-SERT oligomers. Note that, similar to fluorescence correlation experiments, TOCCSL allows only analysis of the mobile fraction of molecules.
We used this experimental strategy to determine the mean aggregation size of mGFP-SERT located at the plasma membrane (Fig. 1b, left) or retained at the ER (Fig. 1b, right) of CHO cells. The differences in the subcellular distributions are apparent in TIRF microscopy; while plasma membranelocalized SERT yields a homogenous intensity distribution over the whole interface of the cell with the glass slide (Fig. 1b, left), we observed the characteristic reticular ER-plasma membrane junctions upon Sar1a-T39N overexpression (Fig. 1b, right). The majority of the protein was freely mobile in both cellular compartments, yielding mobile fractions of 82±8% and 66 ± 12% (s.e.m.; nZ10 cells) for plasma membrane and for the ER in FRAP experiments, respectively.
We first confirmed our recent finding of SERT oligomerization at the plasma membrane: the left panel of Fig. 1b shows the brightness distribution, plotted as a probability density function (pdf) obtained from the TOCCSL images. A large spread in the oligomer distribution was observed, and the mean oligomeric size did not depend on SERT surface density (Fig. 1c, left). The results were strikingly different, when we determined SERT oligomerization at the ER membrane. While the overall oligomer distribution remained highly heterogeneous (Fig. 1b, right), we found a pronounced increase of SERT oligomer size with increasing mean SERT density at the ER (Fig. 1c, right).
A second hallmark of dynamic size equilibration would be the exchange of subunits. To discriminate between stable association and rapid subunit exchange, we used the previously established method of repetitive TOCCSL runs 7 (Fig. 2a). We performed one run per minute (each consisting of a single bleaching pulse and a single recovery image) over 10 min on the very same cell. Pooling data from multiple cells provided brightness distributions as a function of time. By this procedure, the amount of active fluorophores per cell was substantially reduced to about 50%. In this approach, stable interaction of subunits would reduce the total number of observed spots, but would not alter the brightness distribution (Fig. 2a, scenario i). If the exchange rate of subunits was high, however, bleached subunits would mix with unbleached subunits. Over time, this mixing would increase the number of complexes containing both dark and fluorescent subunits, thereby shifting the observable oligomeric distribution towards smaller structures (Fig. 2a, scenario ii). At the plasma membrane, we observed no change in the oligomeric state with increasing number of TOCCSL runs. We thereby confirmed our previous results which indicated stability of SERT oligomers at the minutes (c) At the plasma membrane, the mean oligomeric size is independent from the density of SERT (n422 cells per datapoint; plotted protein densities were 29±17 (s.e.m.) mm À 2 , 402±31 mm À 2 and 840±56 mm À 2 ; s.e.m. of the mean oligomeric sizes were smaller than 0.05). In contrast, at the ER higher expression levels correlate with larger oligomeric sizes (n419 cells per datapoint; plotted protein densities are 153 ± 13 (s.e.m.) mm À 2 , 185 ± 24 mm À 2 , 343±37 mm À 2 and 643±36 mm À 2 ; s.e.m. of the mean oligomeric sizes were smaller than 0.05).  Fig. 2b). At the ER membrane, however, we found a substantial shift towards oligomers with smaller amounts of active fluorophores, indicating rapid exchange of subunits between SERT oligomers (Fig. 2c).
Availability of PIP 2 affects SERT oligomerization. Given the described SERT-PIP 2 interaction 20 and the strong difference of PIP 2 levels between plasma membrane 17 and ER 18 , we hypothesized that PIP 2 impacts on SERT oligomerization. We measured SERT oligomerization at the plasma membrane after depleting PIP 2 levels via activation of phospholipase Cg (PLCg) using the direct PLCg-activator m-3M3FBS (ref. 28). PIP 2 depletion had no influence on the mean mGFP-SERT surface density ( Supplementary Fig. 1). For low SERT surface densities (25 molecules mm À 2 ), we observed a marked shift of SERT oligomers towards smaller complexes, whereas application of the inert orthologue o-3M3FBS did not elicit any effect (38 molecules mm À 2 , Fig. 3a). At higher SERT densities, however, oligomers increased in size (Fig. 3b), indicating rapid equilibration of the oligomerization reaction.
A consequence of rapid equilibration would be the continuous exchange of subunits between SERT oligomers. Hence, to test for the stability of oligomers we performed repetitive TOCCSL runs after incubating cells with m-3M3FBS. The quaternary arrangement of SERT in oligomers now showed rapid subunit exchange (Fig. 3c), which indicated that SERT oligomers were indeed liberated from kinetic trapping. Together, these results show that PIP 2 depletion results in equilibration of SERT oligomerization and concomitant subunit exchange at the plasma membrane. Of note, SERT oligomerization at the plasma membrane lacking PIP 2 resembles the oligomerization behaviour at the ER membrane (Fig. 2). PIP 2 binds directly to SERT and mediates oligomerization. Next, we investigated whether direct PIP 2 binding to SERT sustained oligomer formation or whether PIP 2 would exert an indirect effect due to the downstream metabolites IP 3 or DAG. Recently, we identified the amino acids K352 and K460 as crucial residues for PIP 2 binding to SERT 20 (Fig. 4a); mutation of both residues to alanine yielded a substantial decrease of PIP 2 -induced effects on parachloroamphetamine-induced, SERT-mediated current and efflux. Most importantly, the PIP 2 -SERT interaction was shown to be greatly reduced in pull-down experiments 20 . We used an mGFP-fusion construct of this mutant to evaluate the effect of reduced PIP 2 binding on the oligomeric state. The mGFP-tagged SERT-K352A-K460A double mutant was efficiently trafficked to the plasma membrane and showed similar uptake activity as the wild type 20 . Singlemolecule brightness analysis yielded an oligomeric distribution that differed from wild-type SERT (Fig. 4b): the dominant species are monomers and dimers, while the fraction of trimers and tetramers was reduced to almost baseline levels. The double mutant showed a pronounced density dependence of its oligomeric assembly, which seemed to saturate at a level of B2.8 transporter molecules per oligomer (Fig. 4c). Repetitive TOCCSL runs revealed rapid protomer exchange (Fig. 4d). Together, mGFP-SERT K352A-K460A behaved similar as wildtype SERT after PIP 2 depletion.

Discussion
Although there is a wealth of data supporting the existence of neurotransmitter transporters in oligomeric quaternary structures 10 , the nature of the interaction between the subunits has yet not been unravelled. Here, we examined the size and stability of oligomeric complexes of SERT at two different subcellular localizations, the ER membrane and the plasma membrane. We found that dynamic equilibration of SERT oligomers occurs at the ER membrane. After trafficking through the secretory pathway, the pre-formed oligomers undergo kinetic trapping at the plasma membrane. Pre-equilibration of subunit binding at the ER membrane and kinetic trapping of oligomerized protomers at the plasma membrane enables the cell to spatially decouple the oligomerization process from the final site of oligomer operation.
This appears crucial to render the degree of oligomerization insusceptible to different SERT concentrations at various localizations on the plasma membrane.
Our data indicate that the phosphoinositide PIP 2 plays an essential role in this process and that the different PIP 2 concentrations of the ER membrane and the plasma membrane are responsible for the pronounced differences. While other phosphoinositides would also be plausible candidates for mediating charge-induced SERT oligomerization, some lines of evidence indicate the specific role of PIP 2 in this process. First, PLCg is a highly specific enzyme for PIP 2 hydrolysis, leaving other phosphoinositides virtually unaffected 29 . Experiments shown in Fig. 3 hence reveal the specific contribution of PIP 2 to SERT oligomerization. Second, PIP 2 is by far the most common phosphoinositide in the plasma membrane 16 , thereby outcompeting other phosphoinositide species by shear concentration effects. Third, due to rapid dephosphorylation the ER membrane contains phosphatidylinositol (PI) and phosphatidylinositol(4)phosphate (PI(4)P), but virtually no PIP 2 or other multi-phosphorylated phosphoinositides 16 . Figures 1 and 2 thus render a role of PI and PI(4)P in SERT oligomerization unlikely.
In the following, we propose a model how PIP 2 may cause kinetic trapping of SERT oligomers at the plasma membrane (Fig. 5), which has three cornerstones: . This led to a marked shift of SERT complex sizes towards monomers (dark grey bars). As a negative control, incubation with the inert orthologue o-3M3FBS did not yield any effect (light grey bars) in comparison to the untreated cells (white bars) (n420 cells per experimental condition). SERT surface densities were similar: 25 ± 14 (s.e.m.) mm À 2 (dark grey bars), 38 ± 22 mm À 2 (light grey bars), 29 ± 17 mm À 2 (white bars). (b) PIP 2 depletion via m-3M3FBS resulted in marked dependence of mGFP-SERT oligomerization on mGFP-SERT surface density (n420 cells per datapoint; plotted protein densities are 25±14 (s.e.m.) mm À 2 , 48 ± 24 mm À 2 , 84 ± 17 mm À 2 , 187 ± 28 mm À 2 and 501 ± 40 mm À 2 ; s.e.m. of mean oligomeric sizes were smaller than 0.05). (i) two negatively charged phosphate groups on PIP 2 bind electrostatically to positively charged patches on the cytosolic face of two SERT molecules SERT contains a patch with a strong positive electrostatic potential on the intracellular face which is in contact with the polar headgroups of the membrane 20 (Fig. 4b). The cationic patch is generated by basic amino acid residues (including K352 and K460). The inositol sugar ring of PIP 2 has two functional PO 4 À 2 groups in position 4 and 5, each harbouring a negative charge of À 2 (ref. 30). These functional PO 4 À 2 groups are oriented towards opposing ends of the inositol ring and would therefore have the possibility to interact with two separate SERT monomers. Thereby, they would effectively act as an ionic bridge between the two SERT transporters.
(ii) abrogation of PIP 2 binding leads to rapid equilibration of the oligomerization process The oligomerization process of SERT rapidly equilibrates under conditions, where PIP 2 does not contribute to the subunit association process. This was inferred from the results of three different approaches: the depletion of PIP 2 by enzymatic conversion to IP 3 and diacylglycerol by activation of PLCg (Fig. 3); the retention of SERT in the ER, where PIP 2 levels are extremely low 31 (Figs 1c and 2c); the reduction of PIP 2 binding by introduction of two point mutations 20 (Fig. 4). All conditions revealed qualitatively similar behaviour, that is, the mean size of SERT oligomers became dependent on SERT surface density, and protomers exchanged rapidly between different oligomers. Intrinsic low affinity protein-protein interactions between SERT protomers seem to mediate this process, however, contributions of low PIP 2 concentrations cannot be ruled out. Of note, equilibration of receptor oligomerization at the ER membrane was recently reported for a GPCR 32 .
At this stage, we do not know at which exact subcellular location oligomerization is ultimately adjusted; low PIP 2 levels in virtually all subcellular membranes except for the plasma membrane suggest that the exchange of subunits remains rapid until the fusion of the cargo vesicles with the plasma membrane.
(iii) elevated PIP 2 levels saturate PIP 2 binding sites on SERT, thereby stabilizing the oligomer In principle, PIP 2 binding to the polybasic patch at the cytosolic face of SERT, which includes the lysine residues in positions 352 and 460, increases the affinity to other, undecorated SERT protomers. Naturally occurring concentrations of PIP 2 at the plasma membrane (B20,000-60,000 molecules mm À 2 ; (ref. 33)), however, lead to saturation of SERT with PIP 2 , thereby imposing a charge-based repulsive interaction which precludes further oligomerization.
In consequence, over time most oligomers would disassemble to monomeric SERT until equilibrium is reached, where virtually only PIP 2 -decorated monomers would be present. Importantly, however, there are mechanisms which strongly slow down this equilibration process ('kinetic trapping'). In fact, the high PIP 2 concentration itself may account for such a mechanism. Following this line of argumentation, SERT disassembly may require the complete dissociation of one or several PIP 2 molecules. The high PIP 2 concentrations at the plasma membrane would result in immediate replenishment of the vacant position before separation of the individual protomers.
Hence, according to our model PIP 2 levels are not responsible for tuning the oligomeric distribution of SERT, but instead determine the kinetics for reaching the equilibrium of the oligomerization process: at low PIP 2 levels (as present at the ER membrane) equilibration is fast, whereas at high PIP 2 levels (as present at the plasma membrane) equilibration is substantially slowed down.
In summary, our data show two important steps in the oligomerization of a transmembrane protein: (i) pre-equilibration of subunit binding at the ER membrane and (ii) kinetic trapping of oligomerization at the plasma membrane. By this, the oligomerization process becomes spatially decoupled from the final site of oligomer operation. This could be important to make the degree of oligomerization insusceptible to different SERT concentrations at various locations on the plasma membrane. Coating of glass slides. Proper attachment of the cell lines was ensured by coating the glass slides with fibronectin (Invitrogen) as follows: the slides were cleaned in 70% ethanol supplemented with 2% hydrochloric acid for 15 min and washed three times for 5 min in dH 2 O. 90 ml fibronectin (50 mg ml À 1 in 1 Â PBS) was uniformly distributed on the glass and dried at 50°C. Unbound fibronectin was removed by washing the glass slides three times with 1 Â PBS (PAA Laboratories) before use. PIP 2 depletion experiments were performed on glass slides coated with poly-D-lysine (PDL, Sigma Aldrich). Cleaned slides were incubated with 0.1 mg ml À 1 PDL for 1 h at 37°C and washed three times before use.

Microscopy.
A 488 nm laser (SAPPHIRE HP, Coherent Inc.) was mode-cleaned using a pinhole and the illumination intensity and timing were adjusted with an acousto-optical modulator (model 1205, Isomet) using a custom written software (Labview, National Instruments). The laser beam was focused onto the back-focal plane of a TIRF objective (NA 1.46, Â 100 a Plan APOCHROMAT, Zeiss) mounted on an inverted Zeiss Axiovert 200 microscope. The emission light was filtered using appropriate filter sets for GFP and imaged with a back-illuminated liquid nitrogen cooled CCD camera (Micro Max 1300-PB, Roper Scientific). To restrict the excitation and photobleaching area an adjustable slit aperture (Zeiss) was used as field stop.
All experiments were performed at room temperature. Imaging during all experiments was performed using an objective-type TIRF excitation with an excitation power of B0.5-0.8 kW cm À 2 (determined in epi-configuration) and stroboscopic illumination with excitation times of 3 ms.
Fluorescence recovery after photobleaching. To determine the mobile fraction of mGFP-SERT, an B7 Â 7 mm area of the bottom plasma membrane or the plasma membrane-proximal ER was irreversibly photobleached, and the fluorescence recovery over time was monitored (n410 cells). Photobleaching and readout were performed in TIRF configuration. Data were analysed using in-house algorithms implemented in Matlab. The central part of the bleached region was evaluated by integrating all counts and normalizing to the pre-bleach image. For calculation of the mobile fraction a of mGFP-SERT the resulting curve was fitted with I I0 ¼ aÂð1 À expð t tD ÞÞ.
TOCCSL. TOCCSL experiments were performed as follows ( Supplementary Fig. 2). A pre-bleach image was recorded, which was used for determination of the SERT surface density. After a t pre ¼ 50 ms, a confined region of the cell membrane was photobleached for t bleach ¼ 600-800 ms with a high laser power of B 5-7 kW cm À 2 . To check for complete bleaching, a post-bleach image was recorded t post ¼ 40 ms after the bleach pulse. Finally, the TOCCSL image was recorded after an adjustable recovery time of t recovery ¼ 1,500-12,000 ms. Images were acquired at low excitation power of B0.5-0.8 kW cm À 2 (all excitation intensities were determined in epiconfiguration).
Brightness analysis. For single-molecule analysis, images were analysed using in-house algorithms implemented in MATLAB (MathWorks). Individual diffraction-limited signals were selected and fitted with a Gaussian intensity profile. The fitting routine yielded the single spot brightness B, which was used to determine the oligomeric state of SERT 7,26,27,37 . The obtained brightness values of each diffraction-limited spot in the TOCCSL image were plotted as a pdf r(B). To obtain the brightness distribution of single mGFP molecules r 1 (B), cells were extensively photobleached, which reduced the amount of active fluorophores to a few molecules per mm 2 , so that each potential mGFP-SERT oligomer contained only one active fluorophore at maximum. By autoconvolution, the monomer brightness distribution was used to calculate the brightness distributions for dimers r 2 (B), trimers r 3 (B) and so on. The overall single spot brightness distribution r(B) was then fitted by a linear combination of r 1 (B), r 2 (B), r 3 (B) and so on: Fitting r(B) with equation (1) yielded the fractions a N of the different numbers of co-diffusing active mGFP molecules (with the number of mGFP molecules; Supplementary Fig. 3). Note that a N is proportional to the oligomeric size of SERT, but-due to incomplete mGFP maturation-it slightly underestimates the degree of SERT oligomerization 38 .
At least 750 datapoints were used for n-mer calculations. Using simulation approaches, this sample size was shown to be sufficient for obtaining statistically significant results 39 . To determine error bars, we performed a bootstrapping analysis. Briefly, randomly chosen subsamples containing 50% of the data were analysed using equation (1); shown error bars represent the obtained s.d. from 100 repetitions for each oligomeric size divided by ffiffi ffi 2 p . Mean oligomeric sizes were determined by N mean ¼ Repetitive TOCCSL experiments. To study oligomer stability, a repetitive TOCCSL protocol was applied. One TOCCSL run per minute was performed on the same region of each cell, following the timing protocol shown in Supplementary Fig. 2. Single runs were repeated over 10 min starting from the first bleach pulse. Both bleaching and image acquisition were done in TIRF mode. The TOCCSL image of each run was used for brightness analysis.
Determination of the SERT surface density. The mean fluorescence intensity per mm 2 of the bottom plasma membrane was calculated and divided by the mean single-molecule brightness of mGFP-SERT. To calculate SERT surface densities at plasma membrane-proximal ER, the area fraction of ER-PM junctions was determined from super-resolution images 23 , and the mean intensity of ER-retained SERT was divided by the single-molecule brightness and the respective area fraction.
Molecular modelling and simulations. The crystal structure of human SERT (PDB ID: 5I6X) 40 was used as starting structure. The missing side chains were modelled with MODELLER version 9.15 (ref. 41), creating 100 models using the automodel procedure. The best three models, selected according to the DOPE score 42 , were inserted into a 1-palmitoyl-2-oleoyl-sn-glycero-3phosphatidylcholine containing membrane using the membed procedure 43 . The membrane was pre-equilibrated to contain the SERT transporter 44 . The system was electroneutralized and 150 mM NaCl were added. The environment of SERT was equilibrated while position restraining the transporter. SERT was than released by reducing the position restraints on SERT in four steps, applying 1,000, 100, 10 and 1 kJ mol À 1 , respectively, each time simulating for 2.5 ns. Production runs of 100 ns long equilibrium molecular dynamics simulations were carried out with the GROMACS 5.1 MD package 45 using the AMBER force field 46 for the protein and the Berger parameters 47 for the membrane. The system was maintained at 310 K while coupling protein, membrane and solvent independently using the v-rescale thermostat 48 . The pressure was maintained at 1 bar using the weak coupling algorithm, electrostatic interactions were calculated using the smooth particle mesh Ewald method 49 with a 1.0 nm cutoff. Lennard-Jones interactions were evaluated applying a 1.0 nm cutoff. Long range corrections for energy and pressure were applied.
Code availability. The Matlab Source code for TOCCSL analysis is available at https://github.com/schuetzgroup/TOCCSL_analysis. Data availability. Data supporting the findings of this study are available within the article and its Supplementary Information Files and from the corresponding author upon reasonable request. The PDB accession code 5I6X (SERT Structure) was used in this work.