Identification of the potassium-binding site in serotonin transporter.

Clearance of serotonin (5-hydroxytryptamine, 5-HT) from the synaptic cleft after neuronal signaling is mediated by serotonin transporter (SERT), which couples this process to the movement of a Na+ ion down its chemical gradient. After release of 5-HT and Na+ into the cytoplasm, the transporter faces a rate-limiting challenge of resetting its conformation to be primed again for 5-HT and Na+ binding. Early studies of vesicles containing native SERT revealed that K+ gradients can provide an additional driving force, via K+ antiport. Moreover, under appropriate conditions, a H+ ion can replace K+. Intracellular K+ accelerates the resetting step. Structural studies of SERT have identified two binding sites for Na+ ions, but the K+ site remains enigmatic. Here, we show that K+ antiport can drive substrate accumulation into vesicles containing SERT extracted from a heterologous expression system, allowing us to study the residues responsible for K+ binding. To identify candidate binding residues, we examine many cation binding configurations using molecular dynamics simulations, predicting that K+ binds to the so-called Na2 site. Site-directed mutagenesis of residues in this site can eliminate the ability of both K+ and H+ to drive 5-HT accumulation into vesicles and, in patch clamp recordings, prevent the acceleration of turnover rates and the formation of a channel-like state by K+ or H+. In conclusion, the Na2 site plays a pivotal role in orchestrating the sequential binding of Na+ and then K+ (or H+) ions to facilitate 5-HT uptake in SERT.

Breaking the rules of SLC6 transporters: Export of the human creatine transporter-1 from the endoplasmic reticulum is supported by its N-terminus.

Mutations in the human creatine transporter 1 (CRT1/SLC6A8) cause the creatine transporter deficiency syndrome, which is characterized by intellectual disability, epilepsy, autism, and developmental delay. The vast majority of mutations cause protein misfolding and hence reduce cell surface expression. Hence, it is important to understand the molecular machinery supporting folding and export of CRT1 from the endoplasmic reticulum (ER). All other SLC6 members thus far investigated rely on a C-terminal motif for binding the COPII-component SEC24 to drive their ER export; their N-termini are dispensable. Here, we show that, in contrast, in CRT1 the C-terminal ER-export motif is cryptic and it is the N-terminus, which supports ER export. This conclusion is based on the following observations: (i) siRNA-induced depletion of individual SEC24 isoforms revealed that CRT1 relied on SEC24C for ER export. However, mutations of the C-terminal canonical ER-export motif of CRT1 did not impair its cell surface delivery. (ii) Nevertheless, the C-terminal motif of CRT1 was operational in a chimeric protein comprising the serotonin transporter (SERT/SLC6A4) and the C-terminus of CRT1. (iii) Tagging of the N-terminus-but not the C-terminus-with yellow fluorescent protein (YFP) resulted in ER retention. (iv) Serial truncations of the N-terminus showed that removal of ≥51 residues of CRT1 impaired surface delivery, because the truncated CRT1 were confined to the ER. (v) Mutation of P51 to alanine also reduced cell surface delivery of CRT1 and relieved its dependence on SEC24C. Thus, the ER-export motif in the N-terminus of CRT1 overrides the canonical C-terminal motif.

Probing binding and occlusion of substrate in the human creatine transporter-1 by computation and mutagenesis.

In chordates, energy buffering is achieved in part through phosphocreatine, which requires cellular uptake of creatine by the membrane-embedded creatine transporter (CRT1/SLC6A8). Mutations in human slc6a8 lead to creatine transporter deficiency syndrome, for which there is only limited treatment. Here, we used a combined homology modeling, molecular dynamics, and experimental approach to generate a structural model of CRT1. Our observations support the following conclusions: contrary to previous proposals, C144, a key residue in the substrate binding site, is not present in a charged state. Similarly, the side chain D458 must be present in a protonated form to maintain the structural integrity of CRT1. Finally, we identified that the interaction chain Y148-creatine-Na+ is essential to the process of occlusion, which occurs via a "hold-and-pull" mechanism. The model should be useful to study the impact of disease-associated point mutations on the folding of CRT1 and identify approaches which correct folding-deficient mutants.

The cell cycle protein MAD2 facilitates endocytosis of the serotonin transporter in the neuronal soma.

Monoamine transporters retrieve serotonin (SERT), dopamine (DAT), and norepinephrine (NET) from the synaptic cleft. Transporter internalization contributes to the regulation of their surface expression. Clathrin-mediated endocytosis of plasma membrane proteins requires adaptor protein-2 (AP2), which recruits cargo to the nascent clathrin cage. However, the intracellular portions of monoamine transporters are devoid of a conventional AP2-binding site. Here, we identify a MAD2 (mitotic arrest deficient-2) interaction motif in the C-terminus of SERT, which binds the closed conformation of MAD2 and allows for the recruitment of two additional mitotic spindle assembly checkpoint (SAC) proteins, BubR1 and p31comet , and of AP2. We visualize MAD2, BubR1, and p31comet in dorsal raphe neurons, and depletion of MAD2 in primary serotonergic rat neurons decreases SERT endocytosis in the soma. Our findings do not only provide mechanistic insights into transporter internalization but also allow for rationalizing why SAC proteins are present in post-mitotic neurons.

Regulated rapid round trips: Endocytotic cycling of the dopamine transporter shapes motor learning.

The level of dopamine transporters (DATs) in the neuronal plasma membrane shapes learning and motor coordination in mice. Mechanisms underlying the regulated internalization of DAT and its return to the cell surface have been intensively studied in heterologous cells and in neuronal cell bodies. However, whether this cycling also happens in synaptic boutons, or axon terminals, thought to be the major functional site for DAT expression, was an open question that Kearney and colleagues recently addressed in the JBC. They showed that DAT cycling in the presynaptic specialization of dopaminergic neurons is subject to control by a cell-autonomous loop comprising dopamine autoreceptors and metabotropic glutamate receptors. These results should inform future studies in neural development and motor learning.

A mechanism of uncompetitive inhibition of the serotonin transporter.

The serotonin transporter (SERT/SLC6A4) is arguably the most extensively studied solute carrier (SLC). During its eponymous action - that is, the retrieval of serotonin from the extracellular space - SERT undergoes a conformational cycle. Typical inhibitors (antidepressant drugs and cocaine), partial and full substrates (amphetamines and their derivatives), and atypical inhibitors (ibogaine analogues) bind preferentially to different states in this cycle. This results in competitive or non-competitive transport inhibition. Here, we explored the action of N-formyl-1,3-bis (3,4-methylenedioxyphenyl)-prop-2-yl-amine (ECSI#6) on SERT: inhibition of serotonin uptake by ECSI#6 was enhanced with increasing serotonin concentration. Conversely, the KM for serotonin was lowered by augmenting ECSI#6. ECSI#6 bound with low affinity to the outward-facing state of SERT but with increased affinity to a potassium-bound state. Electrophysiological recordings showed that ECSI#6 preferentially interacted with the inward-facing state. Kinetic modeling recapitulated the experimental data and verified that uncompetitive inhibition arose from preferential binding of ECSI#6 to the K+-bound, inward-facing conformation of SERT. This binding mode predicted a pharmacochaperoning action of ECSI#6, which was confirmed by examining its effect on the folding-deficient mutant SERT-PG601,602AA: preincubation of HEK293 cells with ECSI#6 restored export of SERT-PG601,602AA from the endoplasmic reticulum and substrate transport. Similarly, in transgenic flies, the administration of ECSI#6 promoted the delivery of SERT-PG601,602AA to the presynaptic specialization of serotonergic neurons. To the best of our knowledge, ECSI#6 is the first example of an uncompetitive SLC inhibitor. Pharmacochaperones endowed with the binding mode of ECSI#6 are attractive, because they can rescue misfolded transporters at concentrations, which cause modest transport inhibition.

(+)-Catharanthine and (-)-18-methoxycoronaridine induce antidepressant-like activity in mice by differently recruiting serotonergic and norepinephrinergic neurotransmission.

The antidepressant-like activity of (+)-catharanthine and (-)-18-methoxycoronaridine [(-)-18-MC] was studied in male and female mice using forced swim (FST) and tail suspension tests (TST). The underlying molecular mechanism was assessed by electrophysiological, radioligand, and functional experiments. The FST results showed that acute administration (40 mg/kg) of (+)-catharanthine or (-)-18-MC induces similar antidepressant-like activity in male and female mice at 1 h and 24 h, whereas the TST results showed a lower effect for (-)-18-MC at 24 h. Repeated treatment at lower dose (20 mg/kg) augmented the efficacy of both congeners. The FST results showed that (-)-18-MC reduces immobility and increases swimming times without changing climbing behavior, whereas (+)-catharanthine reduces immobility time, increases swimming times more markedly, and increases climbing behavior. To investigate the contribution of the serotonin and norepinephrine transporters in the antidepressant effects of (+)-catharanthine and (-)-18-MC, we conducted in vitro radioligand and functional studies. Results obtained demonstrated that (+)-catharanthine inhibits norepinephrine transporter with higher potency/affinity than that for (-)-18-MC, whereas both congeners inhibit serotonin transporter with similar potency/affinity. Moreover, whereas no congener activated/inhibited/potentiated the function of serotonin receptor 3A or serotonin receptor 3AB, both increased serotonin receptor 3A receptor desensitization. Depletion of serotonin decreased the antidepressant-like activity of both congeners, whereas norepinephrine depletion only decreased (+)-catharanthine's activity. Our study shows that coronaridine congeners induce antidepressant-like activity in a dose- and time-dependent, and sex-independent, manner. The antidepressant-like property of both compounds involves serotonin transporter inhibition, without directly activating/inhibiting serotonin receptors 3, while (+)-catharanthine also mobilizes norepinephrinergic neurotransmission.

Enriched stable 204Pb as tracer at ultra-low levels in clinical investigations.

The potential of enriched Pb (204Pb) was assessed to monitor pathways of trace levels of Pb in the pg range within the human body via isotope pattern variation in situations where natural lead cannot be used as a tracer due to regulatory limitations. Isotope ratio measurements were accomplished by means of (multi-collector) inductively coupled plasma mass spectrometry including a comparison of single and multi-collector ICP-MS for low-level 204Pb assessment. Isotopic pattern results from a blend of a large quantity of the element with a natural isotopic composition and an enriched stable isotope at orders of magnitude lower levels pose a nontrivial analytical problem. Isotope pattern deconvolution was successfully applied as mathematical tool based on multiple linear regressions. The method allowed for deconvolving the isotope pattern from measured isotope ratios without knowing the quantities of different isotope sources incorporated and mixed into the sample at levels of < 1 pg 204Pb/g blood. The objective of this manuscript is to evaluate and summarize the analytical aspects for Pb isotope pattern deconvolution based on the results of a clinical trial, where a 204Pb-enriched isotope tracer was applied to investigate the bioavailability of orally applied Pb along with purified clinoptilolite tuff as potential supplement. This unique approach allows to reduce tracer amounts to harmless levels to human health, which are in accordance with the legal regulative to study enrichment levels of < 0.01% in human blood.

Safety and efficacy of purified clinoptilolite-tuff treatment in patients with irritable bowel syndrome with diarrhea: Randomized controlled trial.

BACKGROUND: Irritable bowel syndrome (IBS) is a highly prevalent gastrointestinal disorder with poor response to treatment. IBS with predominant diarrhea (IBS-D) is accompanied by abdominal pain as well as high stool frequency and urgency. Purified clinoptilolite-tuff (PCT), which is approved by the Food and Drug Administration for use as a dietary supplement with the brand name G-PUR®, has previously shown therapeutic potential in other indications based on its physical adsorption capacity. AIM: To assess whether symptoms of IBS-D can be ameliorated by oral treatment with PCT. METHODS: In this randomized, placebo-controlled, double-blind pilot study, 30 patients with IBS-D diagnosis based on Rome IV criteria were enrolled. Following a 4-wk run-in phase, 14 patients were randomized to receive a 12-wk treatment with G-PUR® (2 g three times daily), and 16 patients received placebo. The relief from IBS-D symptoms as measured by the proportion of responders according to the Subject's Global Assessment (SGA) of Relief was assessed as the primary outcome. For the secondary outcomes, validated IBS-D associated symptom questionnaires, exploratory biomarkers and microbiome data were collected. RESULTS: The proportions of SGA of Relief responders after 12 wk were comparable in both groups, namely 21% in the G-PUR® group and 25% in the placebo group. After 4 wk of treatment, 36% of patients in the G-PUR® group vs 0% in the placebo group reported complete or considerable relief. An improvement in daily abdominal pain was noted in 94% vs 83% (P = 0.0353), and the median number of days with diarrhea per week decreased by 2.4 d vs 0.3 d in the G-PUR® and placebo groups, respectively. Positive trends were observed for 50% of responders in the Bristol Stool Form Scale. Positive trends were also noted for combined abdominal pain and stool consistency response and the Perceived Stress Questionnaire score. Only 64% in the G-PUR® group compared to 86% in the placebo group required rescue medication intake during the study. Stool microbiome studies showed a minor increase in diversity in the G-PUR® group but not in the placebo group. No PCT-related serious adverse events were reported. CONCLUSION: In this randomized, double-blind, placebo-controlled study, the PCT product, G-PUR®, demonstrated safety and clinical benefit towards some symptoms of IBS-D, representing a promising novel treatment option for these patients.

Cooperative Binding of Substrate and Ions Drives Forward Cycling of the Human Creatine Transporter-1.

Creatine serves as an ATP buffer and is thus an integral component of cellular energy metabolism. Most cells maintain their creatine levels via uptake by the creatine transporter (CRT-1, SLC6A8). The activity of CRT-1, therefore, is a major determinant of cytosolic creatine concentrations. We determined the kinetics of CRT-1 in real time by relying on electrophysiological recordings of transport-associated currents. Our analysis revealed that CRT-1 harvested the concentration gradient of NaCl and the membrane potential but not the potassium gradient to achieve a very high concentrative power. We investigated the mechanistic basis for the ability of CRT-1 to maintain the forward cycling mode in spite of high intracellular concentrations of creatine: this is achieved by cooperative binding of substrate and co-substrate ions, which, under physiological ion conditions, results in a very pronounced (i.e. about 500-fold) drop in the affinity of creatine to the inward-facing state of CRT-1. Kinetic estimates were integrated into a mathematical model of the transport cycle of CRT-1, which faithfully reproduced all experimental data. We interrogated the kinetic model to examine the most plausible mechanistic basis of cooperativity: based on this systematic exploration, we conclude that destabilization of binary rather than ternary complexes is necessary for CRT-1 to maintain the observed cytosolic creatine concentrations. Our model also provides a plausible explanation why neurons, heart and skeletal muscle cells must express a creatine releasing transporter to achieve rapid equilibration of the intracellular creatine pool.

Optimizing the Substrate Uptake Rate of Solute Carriers.

The diversity in solute carriers arose from evolutionary pressure. Here, we surmised that the adaptive search for optimizing the rate of substrate translocation was also shaped by the ambient extracellular and intracellular concentrations of substrate and co-substrate(s). We explored possible solutions by employing kinetic models, which were based on analytical expressions of the substrate uptake rate, that is, as a function of the microscopic rate constants used to parameterize the transport cycle. We obtained the defining terms for five reaction schemes with identical transport stoichiometry (i.e., Na+: substrate = 2:1). We then utilized an optimization algorithm to find the set of numeric values for the microscopic rate constants, which provided the largest value for the substrate uptake rate: The same optimized rate was achieved by different sets of numerical values for the microscopic rate constants. An in-depth analysis of these sets provided the following insights: (i) In the presence of a low extracellular substrate concentration, a transporter can only cycle at a high rate, if it has low values for both, the Michaelis-Menten constant (KM) for substrate and the maximal substrate uptake rate (Vmax). (ii) The opposite is true for a transporter operating at high extracellular substrate concentrations. (iii) Random order of substrate and co-substrate binding is superior to sequential order, if a transporter is to maintain a high rate of substrate uptake in the presence of accumulating intracellular substrate. Our kinetic models provide a framework to understand how and why the transport cycles of closely related transporters differ.

Topically administered purified clinoptilolite-tuff for the treatment of cutaneous wounds: A prospective, randomised phase I clinical trial.

In an ageing society, chronic ulcers pose an increasingly relevant healthcare issue associated with significant morbidity and an increasing financial burden. Hence, there is an unmet medical need for novel, cost-effective therapies that improve healing of chronic cutaneous wounds. This prospective, randomised, open-label, phase I trial investigated the safety and tolerability of topically administered purified clinoptilolite-tuff (PCT), mainly consisting of the naturally occurring zeolite-mineral clinoptilolite, in artificial wounds in healthy male volunteers compared to the standard of care (SoC). We found that topically administered PCT was safe for therapeutic application in acute wounds in healthy male volunteers. No significant differences in wound healing or wound conditions were observed compared to SoC-treated wounds. However, we found a significantly higher proportion of CD68-positive cells and a significantly lower proportion of α-smooth muscle actin-positive cells in PCT-treated wounds. Scanning electron microscopy revealed PCT particles in the restored dermis in some cases. However, these did not impede wound healing or clinical symptoms. Hence, purified PCT could represent an attractive, cost-effective wound treatment promoting the process of healing.

Thermal Unfolding of the Human Serotonin Transporter: Differential Effect by Stabilizing and Destabilizing Mutations and Cholesterol on Thermodynamic and Kinetic Stability.

Folding-deficient mutants of solute carrier 6 (SLC6) family members have been linked to human diseases. The serotonin transporter [(SERT)/SLC6A4] is an important drug target in the treatment of depression, anxiety, and obsessive-compulsive disorders and-with structural information in several conformational states-one of the best understood transporters. Here, we surmised that thermal unfolding offered a glimpse on the folding energy landscape of SLC6 transporters. We carried out molecular dynamic (MD) simulations to understand the mechanistic basis for enhanced and reduced stability, respectively, of the thermostabilized variant SERT-Y110A/I291A/T439S, which had previously been used for crystallization of human SERT in the outward-facing state, and of the folding-deficient SERT-P601A/G602A. We also examined the hydrophobic mismatch caused by the absence of cholesterol to explore the contribution of cholesterol to protein stability. When compared with wild type SERT, the thermodynamic and kinetic stability of SERT-Y110A/I291A/T439S was enhanced. In the other instances, changes in these two components were not correlated: the mutations in SERT-P601A/G602A led to a drop in thermodynamic but an increase in kinetic stability. The divergence was even more pronounced after cholesterol depletion, which reduced thermodynamic stability but increased the kinetic stability of wild type SERT to a level comparable to that of SERT-Y110A/I291A/T439S. We conclude that the low cholesterol content of the endoplasmic reticulum facilitates progression of the folding trajectory by reducing the energy difference between folding intermediates and the native state. SIGNIFICANCE STATEMENT: Point mutations in solute carrier 6 (SLC6) family members cause folding diseases. The serotonin transporter [(SERT)/SLC6A4] is a target for antidepressants and the best understood SLC6. This study produced molecular dynamics simulations and examined thermal unfolding of wild type and mutant SERT variants to understand their folding energy landscape. In the folding-deficient SERT-P012A/G602A, changes in kinetic and thermodynamic stability were not correlated. Similarly, cholesterol depletion lowered thermodynamic but enhanced kinetic stability. These observations allow for rationalizing the action of pharmacochaperones.

Rescue of Misfolded Organic Cation Transporter 3 Variants.

Organic cation transporters (OCTs) are membrane proteins that take up monoamines, cationic drugs and xenobiotics. We previously reported novel missense mutations of organic cation transporter 3 (OCT3, SLC22A3), some with drastically impacted transport capabilities compared to wildtype. For some variants, this was due to ER retention and subsequent degradation of the misfolded transporter. For other transporter families, it was previously shown that treatment of misfolded variants with pharmacological and chemical chaperones could restore transport function to a certain degree. To investigate two potentially ER-bound, misfolded variants (D340G and R348W), we employed confocal and biochemical analyses. In addition, radiotracer uptake assays were conducted to assess whether pre-treatment with chaperones could restore transporter function. We show that pre-treatment of cells with the chemical chaperone 4-PBA (4-phenyl butyric acid) leads to increased membrane expression of misfolded variants and is associated with increased transport capacity of D340G (8-fold) and R348W (1.5 times) compared to untreated variants. We herein present proof of principle that folding-deficient SLC22 transporter variants, in particular those of OCT3, are amenable to rescue by chaperones. These findings need to be extended to other SLC22 members with corroborated disease associations.

Genome Mining-Based Discovery of Blenny Fish-Derived Peptides Targeting the Mouse κ-Opioid Receptor.

Over the past years, peptides have attracted increasing interest for G protein-coupled receptor (GPCR) drug discovery and development. Peptides occupy a unique chemical space that is not easily accessible for small molecules and antibodies and provide advantages over these ligand classes such as lower toxicity and higher selectivity. The κ-opioid receptor (KOR) is a prototypic GPCR and an appealing therapeutic target for the development of safer and more effective analgesics. Recently, peptides have emerged as analgesic drug candidates with improved side effect profiles. We have previously identified plant-derived peptides, which activate KOR. Based on this precedent, here we relied on publicly available databases to discover novel KOR peptide ligands by genome mining. Using human preprodynorphin as a query, we identified blenny fish-derived peptides, referred to as blenniorphins, capable of binding to and activating KOR with nanomolar affinity and potency, respectively. Additionally, the blenniorphins altered ß-arrestin-2 recruitment at the KOR. Our study demonstrates the utility of genome mining to identify peptide GPCR ligands with intriguing pharmacological properties and unveils the potential of blenny fishes as a source for novel KOR ligands.

Assessing the Accuracy of Sales Forecasts Submitted by Pharmaceutical Companies Applying for Reimbursement in Austria.

Objectives: Reimbursement decisions on new medicines require an assessment of their value. In Austria, when applying for reimbursement of new medicines, pharmaceutical companies are also obliged to submit forecasts of future sales. We systematically examined the accuracy of these pharmaceutical sales forecasts and hence the usefulness of these forecasts for reimbursement evaluations. Methods: We retrospectively analyzed reimbursement applications of 102 new drugs submitted between 2005 and 2014, which were accepted for reimbursement outside of hospitals, and for which actual reimbursed sales were available for at least 3 years. The main outcome variable was the accuracy ratio, defined as the ratio of forecasted sales submitted by pharmaceutical companies when applying for reimbursement to actual sales from reimbursement data. Results: The median accuracy ratio [95% confidence interval] was 1.33 [1.03; 1.74, range 0.15-37.5], corresponding to a median overestimation of actual sales by 33%. Forecasts of actual sales for 55.9% of all examined products either overestimated actual sales by more than 100% or underestimated them by more than 50%. The accuracy of sales forecasts did not show systematic change over the analyzed decade nor was it discernibly influenced by reimbursement status (restricted or unrestricted), the degree of therapeutic benefit, or the therapeutic area of the pharmaceutical product. Sales forecasts of drugs with a higher degree of innovation and those within a dynamic market tended to be slightly more accurate. Conclusions: The majority of sales forecasts provided by applicants for reimbursement evaluations in Austria were highly inaccurate and were on average too optimistic. This is in line with published results for other jurisdictions and highlights the need for caution when using such forecasts for reimbursement procedures.

Plant-Derived Cyclotides Modulate κ-Opioid Receptor Signaling.

Cyclotides are plant-derived disulfide-rich peptides comprising a cyclic cystine knot, which confers remarkable stability against thermal, proteolytic, and chemical degradation. They represent an emerging class of G protein-coupled receptor (GPCR) ligands. In this study, utilizing a screening approach of plant extracts and pharmacological analysis we identified cyclotides from Carapichea ipecacuanha to be ligands of the κ-opioid receptor (KOR), an attractive target for developing analgesics with reduced side effects and therapeutics for multiple sclerosis (MS). This prompted us to verify whether [T20K]kalata B1, a cyclotide in clinical development for the treatment of MS, is able to modulate KOR signaling. T20K bound to and fully activated KOR in the low µM range. We then explored the ability of T20K to allosterically modulate KOR. Co-incubation of T20K with KOR ligands resulted in positive allosteric modulation in functional cAMP assays by altering either the efficacy of dynorphin A1-13 or the potency and efficacy of U50,488 (a selective KOR agonist), respectively. In addition, T20K increased the basal response upon cotreatment with U50,488. In the bioluminescence resonance energy transfer assay T20K negatively modulated the efficacy of U50,488. This study identifies cyclotides capable of modulating KOR and highlights the potential of plant-derived peptides as an opportunity to develop cyclotide-based KOR modulators.

Concomitant oral intake of purified clinoptilolite tuff (G-PUR) reduces enteral lead uptake in healthy humans.

Lead exposure can cause substantial organ damage. Enteral lead absorption may be reduced by concomitant intake of clinoptilolite tuff, a zeolite from natural sources. This study aimed to assess the effect of purified clinoptilolite tuff (G-PUR) on enteral lead uptake in adults using stable lead isotope 204Pb as a tracer. In this randomized, placebo-controlled, double-blind, parallel-group study, 42 healthy participants were randomized to receive oral G-PUR 2.0 g, 2 * 2.0 g, or placebo, together with 2.5 µg of 204Pb in water. The enrichment of 204Pb caused by the tracer in blood and urine was measured by mass spectrometry. G-PUR was well tolerated. The mean maximum 204Pb enrichment of 0.505% of total blood lead was significantly higher (p < 0.0001) in the placebo group compared to G-PUR 2.0 g (0.073%) or G-PUR 2 * 2.0 g (0.057%) group. Normalized 204Pb AUC0-192 was 86.5, 11.9, and 8.5% * h without and with G-PUR 2.0 g, and G-PUR 2 * 2.0 g, respectively (p < 0.0001 vs. placebo). This smaller 204Pb exposure was paralleled by a reduced urinary excretion in subjects receiving G-PUR. Concomitant oral intake of purified clinoptilolite tuff reduced enteral uptake of 204Pb in healthy humans by approximately 90%. The reduced bioavailability is demonstrable by a decrease of 204Pb tracer enrichment in blood and urine.Trial registration: clinicaltrials.gov identifier: NCT04138693, registered 24/10/2019.

Handling of intracellular K+ determines voltage dependence of plasmalemmal monoamine transporter function.

The concentrative power of the transporters for dopamine (DAT), norepinephrine (NET), and serotonin (SERT) is thought to be fueled by the transmembrane Na+ gradient, but it is conceivable that they can also tap other energy sources, for example, membrane voltage and/or the transmembrane K+ gradient. We have addressed this by recording uptake of endogenous substrates or the fluorescent substrate APP+(4-(4-dimethylamino)phenyl-1-methylpyridinium) under voltage control in cells expressing DAT, NET, or SERT. We have shown that DAT and NET differ from SERT in intracellular handling of K+. In DAT and NET, substrate uptake was voltage-dependent due to the transient nature of intracellular K+ binding, which precluded K+ antiport. SERT, however, antiports K+ and achieves voltage-independent transport. Thus, there is a trade-off between maintaining constant uptake and harvesting membrane potential for concentrative power, which we conclude to occur due to subtle differences in the kinetics of co-substrate ion binding in closely related transporters.

Extracellular loops of the serotonin transporter act as a selectivity filter for drug binding.

The serotonin transporter (SERT) shapes serotonergic neurotransmission by retrieving its eponymous substrate from the synaptic cleft. Ligands that discriminate between SERT and its close relative, the dopamine transporter DAT, differ in their association rate constant rather than their dissociation rate. The structural basis for this phenomenon is not known. Here we examined the hypothesis that the extracellular loops 2 (EL2) and 4 (EL4) limit access to the ligand-binding site of SERT. We employed an antibody directed against EL4 (residues 388-400) and the antibody fragments 8B6 scFv (directed against EL2 and EL4) and 15B8 Fab (directed against EL2) and analyzed their effects on the transport cycle of and inhibitor binding to SERT. Electrophysiological recordings showed that the EL4 antibody and 8B6 scFv impeded the initial substrate-induced transition from the outward to the inward-facing conformation but not the forward cycling mode of SERT. In contrast, binding of radiolabeled inhibitors to SERT was enhanced by either EL4- or EL2-directed antibodies. We confirmed this observation by determining the association and dissociation rate of the DAT-selective inhibitor methylphenidate via electrophysiological recordings; occupancy of EL2 with 15B8 Fab enhanced the affinity of SERT for methylphenidate by accelerating its binding. Based on these observations, we conclude that (i) EL4 undergoes a major movement during the transition from the outward to the inward-facing state, and (ii) EL2 and EL4 limit access of inhibitors to the binding of SERT, thus acting as a selectivity filter. This insight has repercussions for drug development.