Design and synthesis of S-citalopram-imprinted polymeric sorbent: Characterization and application in enantioselective separation.

The current work describes the efficient creation and employment of a new S-citalopram selective polymeric sorbent, made from poly(divinylbenzene-maleic anhydride-styrene). The process began by using suspension polymerization technique in the synthesis of poly(styrene-maleic anhydride-divinylbenzene) microparticles. These were then modified with ethylenediamine, developing an amido-succinic acid-based polymer derivative. The S-citalopram, a cationic molecule, was loaded onto these developed anionic polymer particles. Subsequently, the particles were post-crosslinked using glyoxal, which reacts with the amino group residues of ethylenediamine. S-citalopram was extracted from this matrix using an acidic solution, which also left behind stereo-selective cavities in the S-citalopram imprinted polymer, allowing for the selective re-adsorption of S-citalopram. The attributes of the polymer were examined through methods such as 13C NMR, FTIR, thermogravemetric and elemental analyses. SEM was used to observe the shapes and structures of the particles. The imprinted polymers demonstrated a significant ability to adsorb S-citalopram, achieving a capacity of 878 mmol/g at a preferred pH level of 8. It proved efficient in separating enantiomers of (±)-citalopram via column methods, achieving an enantiomeric purity of 97 % for R-citalopram upon introduction and 92 % for S-citalopram upon release.

Dynamic extracellular vestibule of human SERT: Unveiling druggable potential with high-affinity allosteric inhibitors.

The serotonin transporter (SERT) tightly regulates synaptic serotonin levels and has been the primary target of antidepressants. Binding of inhibitors to the allosteric site of human SERT (hSERT) impedes the dissociation of antidepressants bound at the central site and may enhance the efficacy of such antidepressants to potentially reduce their dosage and side effects. Here, we report the identification of a series of high-affinity allosteric inhibitors of hSERT in a unique scaffold, with the lead compound, Lu AF88273 (3-(1-(2-(1H-indol-3-yl)ethyl)piperidin-4-yl)-6-chloro-1H-indole), having 2.1 nM allosteric potency in inhibiting imipramine dissociation. In addition, we find that Lu AF88273 also inhibits serotonin transport in a noncompetitive manner. The binding pose of Lu AF88273 in the allosteric site of hSERT is determined with extensive molecular dynamics simulations and rigorous absolute binding free energy perturbation (FEP) calculations, which show that a part of the compound occupies a dynamically formed small cavity. The predicted binding location and pose are validated by site-directed mutagenesis and can explain much of the structure-activity relationship of these inhibitors using the relative binding FEP calculations. Together, our findings provide a promising lead compound and the structural basis for the development of allosteric drugs targeting hSERT. Further, they demonstrate that the divergent allosteric sites of neurotransmitter transporters can be selectively targeted.

Nontarget screening based on molecular networking strategy to identify transformation products of citalopram and sertraline in wastewater.

Citalopram (CIT) and sertraline (SER) are highly consumed antidepressants worldwide and have been extensively detected in wastewater. Due to the incomplete mineralization, transformation products (TPs) of them can be detected in wastewater. Comparing with parent compounds, knowledge on TPs are limited. To fill these research gaps, lab-scale batch experiments, WWTPs sampling and in silico toxicity prediction were implemented to investigate the structure, occurrence and toxicity of TPs. Based on molecular networking nontarget strategy, 13 TPs of CIT and 12 TPs of SER were tentatively identified. Among them, 4 TPs from CIT and 5 TPs from SER were newly found in present study. TPs identification results compared with results obtained from previous nontarget strategies demonstrated that the excellent performances for molecular networking strategy on candidate TPs prioritizing and new TPs finding, especially for low abundance TPs. Further, transformation pathways for CIT and SER in wastewater were proposed. Newly identified TPs provided insights on defluorination, formylation and methylation for CIT and dehydrogenation, N-malonylation and N-acetoxylation for SER transformed in wastewater. Nitrile hydrolysis and N-succinylation were found to be the dominant transformation pathways for CIT and SER in wastewater, respectively. WWTPs sampling results shown the concentrations of SER and CIT ranged from 0.46 to 28.66 ng/L and 17.16 to 58.36 ng/L. In addition, 7 TPs of CIT and 2 TPs of SER found in lab-scale wastewater samples were found in WWTPs. In silico results suggested 2 TPs of CIT may be more toxic than CIT toward all three trophic levels organisms. Present study provides new insights into the transformation processes of CIT and SER in wastewater. In addition, the necessity of paying more attention on TPs was further highlighted from the aspects of toxicity for TPs of CIT and SER in effluent of WWTPs.

Allosteric Binding of MDMA to the Human Serotonin Transporter (hSERT) via Ensemble Binding Space Analysis with ΔG Calculations, Induced Fit Docking and Monte Carlo Simulations.

Despite the recent promising results of MDMA (3,4-methylenedioxy-methamphetamine) as a psychotherapeutic agent and its history of misuse, little is known about its molecular mode of action. MDMA enhances monoaminergic neurotransmission in the brain and its valuable psychoactive effects are associated to a dual action on the 5-HT transporter (SERT). This drug inhibits the reuptake of 5-HT (serotonin) and reverses its flow, acting as a substrate for the SERT, which possesses a central binding site (S1) for antidepressants as well as an allosteric (S2) one. Previously, we characterized the spatial binding requirements for MDMA at S1. Here, we propose a structure-based mechanistic model of MDMA occupation and translocation across both binding sites, applying ensemble binding space analyses, electrostatic complementarity, and Monte Carlo energy perturbation theory. Computed results were correlated with experimental data (r = 0.93 and 0.86 for S1 and S2, respectively). Simulations on all hSERT available structures with Gibbs free energy estimations (ΔG) revealed a favourable and pervasive dual binding mode for MDMA at S2, i.e., adopting either a 5-HT or an escitalopram-like orientation. Intermediate ligand conformations were identified within the allosteric site and between the two sites, outlining an internalization pathway for MDMA. Among the strongest and more frequent interactions were salt bridges with Glu494 and Asp328, a H-bond with Thr497, a π-π with Phe556, and a cation-π with Arg104. Similitudes and differences with the allosteric binding of 5-HT and antidepressants suggest that MDMA may have a distinctive chemotype. Thus, our models may provide a framework for future virtual screening studies and pharmaceutical design and to develop hSERT allosteric compounds with a unique psychoactive MDMA-like profile.

Occlusion of the human serotonin transporter is mediated by serotonin-induced conformational changes in the bundle domain.

The human serotonin transporter (hSERT) terminates neurotransmission by removing serotonin (5HT) from the synaptic cleft, an essential process for proper functioning of serotonergic neurons. Structures of the hSERT have revealed its molecular architecture in four conformations, including the outward-open and occluded states, and show the transporter's engagement with co-transported ions and the binding mode of inhibitors. In this study, we investigated the molecular mechanism by which the hSERT occludes and sequesters the substrate 5HT. This first step of substrate uptake into cells is a structural change consisting of the transition from the outward-open to the occluded state. Inhibitors such as the antidepressants citalopram, fluoxetine, and sertraline inhibit this step of the transport cycle. Using molecular dynamics simulations, we reached a fully occluded state, in which the transporter-bound 5HT becomes fully shielded from both sides of the membrane by two closed hydrophobic gates. Analysis of 5HT-triggered occlusion showed that bound 5HT serves as an essential trigger for transporter occlusion. Moreover, simulations revealed a complex sequence of steps and showed that movements of bundle domain helices are only partially correlated. 5HT-triggered occlusion is initially dominated by movements of transmembrane helix 1b, while in the final step, only transmembrane helix 6a moves and relaxes an intermediate change in its secondary structure.

Selective LC-MS/MS determination of citalopram enantiomers and application to a pharmacokinetic evaluation of generic and reference formulations.

Two methods using LC-MS/MS were validated to quantify citalopram (CTP) racemate [(R/S)-CTP] and the enantiomers (R)-CTP and (S)-CTP in human plasma, respectively. Paroxetine hydrochloride was used as the internal standard, and samples were extracted by protein precipitation with acetonitrile. The non-enantioselective method was conducted using a C18 column, and the mobile phase consisted of water for solvent A and acetonitrile for solvent B, both with 0.1% formic acid. For the chiral method, an analytical column Lux Cellulose-1 was used. Mobile phase A was composed of water with 0.025% of formic acid and 0.05% of diethylamine, and mobile phase B consisted of acetonitrile:2-propanol (95:5, v/v). No significant matrix effects were observed at the retention times of analytes and internal standard. The mean recovery was 89%, and the assays were linear in the concentration range of 1-50 and 5-30 ng/mL for the non-enantioselective and enantioselective methods, respectively. The intra- and inter-day precisions of both methods were less than 12.30%, and the accuracies were less than 12.13%. The validated methods were successfully applied to a pharmacokinetic study in which 20-mg CTP tablets were administered to healthy volunteers, and their plasma levels were monitored over time in a bioequivalence study. HIGHLIGHTS: Simple and rapid LC-MS/MS method for the quantification of citalopram and its enantiomers in human plasma. Both methods were demonstrated to be selective, reliable, and sensitive. Both methods have sufficient sensitivity to quantify the steady state through concentrations already reported for citalopram and escitalopram. Validated method presented in this study can be suitably applied to pharmacokinetic studies involving citalopram and escitalopram. Bland-Altman analysis suggested that non-enantioselective and enantioselective methods can be applied in pharmacokinetic studies.

Investigation of the Aquatic Photolytic and Photocatalytic Degradation of Citalopram.

This study investigated the direct and indirect photochemical degradation of citalopram (CIT), a selective serotonin reuptake inhibitor (SSRI), under natural and artificial solar radiation. Experiments were conducted in a variety of different operating conditions including Milli-Q (MQ) water and natural waters (lake water and municipal WWT effluent), as well as in the presence of natural water constituents (organic matter, nitrate and bicarbonate). Results showed that indirect photolysis can be an important degradation process in the aquatic environment since citalopram photo-transformation in the natural waters was accelerated in comparison to MQ water both under natural and simulated solar irradiation. In addition, to investigate the decontamination of water from citalopram, TiO2-mediated photocatalytic degradation was carried out and the attention was given to mineralization and toxicity evaluation together with the identification of by-products. The photocatalytic process gave rise to the formation of transformation products, and 11 of them were identified by HPLC-HRMS, whereas the complete mineralization was almost achieved after 5 h of irradiation. The assessment of toxicity of the treated solutions was performed by Microtox bioassay (Vibrio fischeri) and in silico tests showing that citalopram photo-transformation involved the formation of harmful compounds.

Gelatin microsphere coated Fe3O4@graphene quantum dots nanoparticles as a novel magnetic sorbent for ultrasound-assisted dispersive magnetic solid-phase extraction of tricyclic antidepressants in biological samples.

Gelatin microsphere-coated Fe3O4@graphene quantum dots (Fe3O4@GQD@GM) were designed and synthesized as a novel sorbent via ultrasonic-assisted dispersive magnetic solid-phase extraction (UA-DMSPE) method. The synthesized sorbent was identified and confirmed by FT-IR, XRD, VSM, and SEM techniques. UA-DMSPE was combined with corona discharge ion mobility spectrometry for trace determination of desipramine, sertraline, and citalopram. Effective parameters were considered and optimized. The proposed method, under optimal conditions, showed excellent linearity in different concentration ranges (2-700 ng mL-1, R2 > 0.995), repeatability (RSD < 5.1%), good sensitivity (LODs in the range 0.6-1.5 ng mL-1), high preconcentration factor (PF = 207-218), and acceptable relative recoveries (93.5-101.8%). Eventually, this method was used to determine tricyclic antidepressants in various biological samples. Schematic presentation of the microextraction and monitoring of TCAs by ultrasonic-assisted dispersive magnetic solid phase microextraction-ion mobility spectrometry producer.

Capillary Electrophoresis Method for Determination of Escitalopram Oxalate in Urine Samples and Different Dosage Forms.

Application of capillary electrophoresis (CE) has become a rapidly growing analytical technique for the estimation of drugs in pharmaceutical dosage forms and biological fluids. In this study, an effective and sensitive method was developed for the determination of escitalopram oxalate (ESC-OX) by CE with diode-array detection at 200 nm. The separation was achieved by a fused silica capillary with 40 cm effective length (48.5 cm total, 75 µm i.d.). The background electrolyte was composed of 15 mM phosphate buffer (pH 2.5). The applied potential was 22.5 kV, and the samples were injected at 50 mbar pressure for 10 s. Metoprolol was used as an internal standard (IS). The migration time under these optimum conditions was 6.51 ± 0.07 and 6.73 ± 0.08 min for ESC-OX and IS, respectively, with total run time 7 min. The method was validated for linearity, precision, accuracy, specificity and sensitivity. The limit of detection was calculated as 3.85 and 5.07 ng mL-1 for standard and urine samples, respectively. The developed method was employed successfully for the determination of ESC-OX in different pharmaceutical dosage forms and spiked human urine after simple liquid-liquid extraction with good recovery.

Identification and characterization of citalopram new metabolites with the use of UHPLC-Q-TOF technique: In silico toxicity assessment of the identified transformation products.

In this study the metabolite profiling of citalopram with the use of human liver microsomes as well as the complementary photocatalytic method were established. This strategy allowed the detection of five metabolites of citalopram including 3-hydroxycitalopram and 3-oxocitalopram which were found as a new and not previously described metabolites of this drug The photocatalytic simulation of metabolism was carried out using tungsten (VI) oxide nanopowders with the different particle sizes, which allowed to examine the effect of this photocatalyst parameter on the mapping of metabolic processes. The accurate characterization of all observed structures was possible due to the use of ultra-high-pressure liquid chromatography and high-resolution mass spectrometry combined system as a highly useful technique in drug metabolism studies. In order to perform the toxicity prediction of citalopram and its metabolites, the acute toxicity to rodents, as well as genotoxicity, carcinogenicity, developmental toxicity and receptor-mediated toxicity was calculated basing on the in silico tools.

The mechanism of a high-affinity allosteric inhibitor of the serotonin transporter.

The serotonin transporter (SERT) terminates serotonin signaling by rapid presynaptic reuptake. SERT activity is modulated by antidepressants, e.g., S-citalopram and imipramine, to alleviate symptoms of depression and anxiety. SERT crystal structures reveal two S-citalopram binding pockets in the central binding (S1) site and the extracellular vestibule (S2 site). In this study, our combined in vitro and in silico analysis indicates that the bound S-citalopram or imipramine in S1 is allosterically coupled to the ligand binding to S2 through altering protein conformations. Remarkably, SERT inhibitor Lu AF60097, the first high-affinity S2-ligand reported and characterized here, allosterically couples the ligand binding to S1 through a similar mechanism. The SERT inhibition by Lu AF60097 is demonstrated by the potentiated imipramine binding and increased hippocampal serotonin level in rats. Together, we reveal a S1-S2 coupling mechanism that will facilitate rational design of high-affinity SERT allosteric inhibitors.

Development and pharmaceutical evaluation of oral fast dissolving thin film of escitalopram: A patient friendly dosage form.

The current study focused on the development, optimization and pharmaceutical evaluation of a mouth dissolving film of Escitalopram 5mg. The designing and optimization of the formulations have been carried out through Design-Expert ⌖ Ver. 9, using central composite response surface methodology. The software generated six optimized formulations have been fabricated via solvent casting method incorporated with HPMC and PEG 400 as Plasticizer. The developed formulations were assessed for various quality attributes including weight variation, drug-excipients interaction, dryness/ tack test, thickness, percent elongation, swelling index, disintegration, folding endurance, surface pH, content uniformity, assay, moisture uptake, stability, and organoleptic properties. A validated spectrophotometric method has been used to ascertain drug content. The formulations were subjected for stability studies for six months in accordance with ICH accelerated stability studies guidelines. No stability issue has been observed. All the test formulations have shown satisfactory in vitro release of escitalopram whereas most promising results have been exhibited by F5 and F6 formulations. The study concluded that a unique, novel, safe and stable formulation of oral fast dissolving thin films of escitalopram can be formulated with ease. The preparation method was simple and reproducible with scale-up tendency so that it can fulfill the need of the commercial manufacturing process.

Maltodextrin-modified graphene oxide for improved enantiomeric separation of six basic chiral drugs by open-tubular capillary electrochromatography.

An electrochromatographic capillary was modified with graphene oxide (GO), and the coating was characterized by scanning electron microscopy, energy dispersive X-ray spectrometry, and Fourier transform infrared spectra. By utilizing maltodextrin (MD) as the chiral selector, the basic chiral drugs nefopam (NEF), amlodipine (AML), citalopram hydrobromide (CIT), econazole (ECO), ketoconazole (KET) and cetirizine hydrochloride (CET) can be enantiomerically separated on this CEC. Compared with an uncoated silica capillary, the resolutions are markedly improved (AML: 0.32 → 1.45; ECO: 0.55 → 1.89; KET: 0.88 → 4.77; CET: 0.81 → 2.46; NEF: 1.46 → 2.83; CIT: 1.77 → 4.38). Molecular modeling was applied to demonstrate the mechanism of enantioseparation, which showed a good agreement with the experimental results. Graphical abstractSchematic representation of the preparation of graphene oxide-modified capillary (GO@capillary) for enantioseparation of drug enantiomers. The monolayered GO was used as the coating of the GO@capillary. Then the capillary was applied to construct capillary electrochromatography system with maltodextrin for separation of basic chiral drugs.

Oxidation of Citalopram with Sodium Hypochlorite and Chlorine Dioxide: Influencing Factors and NDMA Formation Kinetics.

The highly prescribed antidepressant, citalopram, as one of newly emerging pollutants, has been frequently detected in the aquatic environment. Citalopram oxidation was examined during sodium hypochlorite (NaOCl) and chlorine dioxide (ClO2) chlorination processes since conventional wastewater treatment plants cannot remove citalopram effectively. Citalopram has been demonstrated to form N-nitrosodimethylamine (NDMA) during chlorination in our previous study. Further investigation on NDMA formation kinetics was conducted in the present study. Influences of operational variables (disinfectant dose, pH value) and water matrix on citalopram degradation, as well as NDMA generation, were evaluated. The results indicated high reactivity of citalopram with NaOCl and ClO2. NDMA formation included two stages during CIT oxidation, which were linear related with reaction time. NaOCl was more beneficial to remove CIT, but it caused more NDMA formation. Increasing disinfectant dosage promoted citalopram removal and NDMA formation. However, no consistent correlation was found between citalopram removal and pH. Contrary to the situation of citalopram removal, NDMA generation was enhanced when citalopram was present in actual water matrices, especially in secondary effluent. DMA, as an intermediate of citalopram chlorination, contributed to NDMA formation, but not the only way.

Conformational dynamics of the human serotonin transporter during substrate and drug binding.

The serotonin transporter (SERT), a member of the neurotransmitter:sodium symporter family, is responsible for termination of serotonergic signaling by re-uptake of serotonin (5-HT) into the presynaptic neuron. Its key role in synaptic transmission makes it a major drug target, e.g. for the treatment of depression, anxiety and post-traumatic stress. Here, we apply hydrogen-deuterium exchange mass spectrometry to probe the conformational dynamics of human SERT in the absence and presence of known substrates and targeted drugs. Our results reveal significant changes in dynamics in regions TM1, EL3, EL4, and TM12 upon binding co-transported ions (Na+/K+) and ligand-mediated changes in TM1, EL3 and EL4 upon binding 5-HT, the drugs S-citalopram, cocaine and ibogaine. Our results provide a comprehensive direct view of the conformational response of SERT upon binding both biologically relevant substrate/ions and ligands of pharmaceutical interest, thus advancing our understanding of the structure-function relationship in SERT.

α-Fluorination of carbonyls with nucleophilic fluorine.

Given the unique properties of fluorine, and the ability of fluorination to change the properties of organic molecules, there is significant interest from medicinal chemists in innovative methodologies that enable the synthesis of new fluorinated motifs. State-of-the-art syntheses of α-fluorinated carbonyl compounds invariably rely on electrophilic fluorinating agents, which can be strongly oxidizing and difficult to handle. Here we show that reversing the polarity of the enolate partner to that of an enolonium enables nucleophilic fluorinating agents to be used for direct chemoselective α-C-H-fluorination of amides. Reduction of these products enables facile access to ß-fluorinated amines and the value of this methodology is shown by the easy preparation of a number of fluorinated analogues of drugs and agrochemicals. A fluorinated analogue of citalopram, a marketed antidepressant drug, is presented as an example of the preserved biological activity after fluorination.

Transformation products of citalopram: Identification, wastewater analysis and in silico toxicological assessment.

The objective of this study was to identify transformation products (TPs) of citalopram (CIT), an antidepressant drug, in laboratory experiments. Moreover, toxicity predictions and analyzes in wastewater samples were performed. For the formation of TPs, raw water was used for the processes of hydrolysis; photodegradation under ultraviolet (UV) irradiation and chlorination. The toxicities were predicted by computational toxicity assessment. The TPs were identified by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) in broadband collision induced dissociation (bbCID) acquisition mode and product ion scan mode (MS/MS). The probable structures of the TPs under study were established based on accurate mass, fragmentations observed in the MS spectra and prediction tools software. The experiments resulted in seventeen possible identified TPs and their stability and formation was monitored over time in the experiments. Two of these TPs were identified in wastewater samples It was also observed that most of TPs formed were either less toxic then CIT or had a similar toxicity.

Investigation of dextrin-based synergistic system with chiral ionic liquids as additives for enantiomeric separation in capillary electrophoresis.

In this paper, two spiral structure CILs, 1-butyl-3-methylimidazolium(T-4)-bis[(2S)-2-(hydroxy-κO)-3-methyl-butanoato-κO]borate(BMIm+BLHvB-) and 1-butyl-3-methylimidazolium (T-4)-bis[(αS)-α-(hydroxy-κO)-4-methyl-benzeneacetato-κO]borate (BMIm+BSMB-)were applied to evaluate their potential synergistic effect with dextrin for CE enantiomeric separation. The established dextrin-based synergistic system with CILs as additives showed good separation performance towards four tested drugs, including duloxetine, ketoconazole, sulconazole and citalopram. It was also observed that significantly improved separation and selectivity for tested analytes were achieved in CILs/dextrin synergistic system compared to single dextrin system. Primary parameters, such as the concentration of CIL, dextrin concentration, buffer pH and applied voltage, were systematically investigated to optimize the enantiomeric separation with BMIm+BLHvB-/dextrin as model system. Finally, the method of Statistical Product and Service Solutions (SPSS) was exploited to further elucidate the influence of experimental parameters on the synergistic effect.

Computational characterization of the selective inhibition of human norepinephrine and serotonin transporters by an escitalopram scaffold.

Human norepinephrine and serotonin transporters (hNET and hSERT) are closely related monoamine transporters (MATs) that regulate neurotransmitter signaling in neurons and are primary targets for a wide range of therapeutic drugs used in the treatment of mood disorders. The subtle modifications of an escitalopram scaffold exhibit distinct selective inhibition profiles of hNET and hSERT. However, the structural details of escitalopram scaffold binding to hSERT and (or) hNET are poorly understood and still remain a great challenge. In this work, on the basis of more recently solved X-ray crystallographic structure of hSERT in complex with escitalopram, 3 µs long all-atom MD simulations and binding free energy calculations via MM/GB(PB)SA, thermodynamic integration (TI) and MM/3D-RISM methods were performed to reproduce experimental free energies. And both MM/GBSA and TI have a high correlation coefficient (R2 = 0.95 and 0.96, respectively) between the relative binding free energies of the calculated and experimental values. Furthermore, MM/GBSA per-residue energy decomposition, molecular interaction fingerprints and thermodynamics-structure relationship analysis were employed to investigate and characterize the selectivity of the escitalopram scaffold with three modifications (escitalopram, ligand10 and talopram) to hNET and hSERT. As a result, 4 warm spots (A73, Y151, A477 and I481) in hNET and 4 warm spots (A96, A173, T439 and L443) in hSERT were thus discovered to exert a pronounced effect on the selective inhibition of hNET and hSERT by the studied ligands. These simulation results would provide great insight into the design of inhibitors with the desired selectivity to hNET and hSERT, thus further promoting the research of more efficacious antidepressants.

Two Ligand Binding Sites in Serotonin Transporter Revealed by Nanopharmacological Force Sensing.

The number of ligand binding sites in neurotransmitter-sodium symporters has been determined by crystal structure analysis and molecular pharmacology with controversial results. Here, we designed molecular tools to measure the interaction forces between the serotonin transporter (SERT) and S-citalopram on the single-molecule level by means of atomic force microscopy. Force spectroscopy allows for the extraction of dynamic information under physiological conditions which is inaccessible via X-ray crystallography. Two populations of distinctly different binding strength between S-citalopram and SERT were demonstrated in Na+-containing buffer. In Li+-containing buffer, SERT showed merely low-force interactions, whereas the vestibular mutant SERT-G402H only displayed the high force population. These observations provide physical evidence for the existence of two different binding sites in SERT when tested under near-physiological conditions.