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.

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.

Allosteric Inhibition and Pharmacochaperoning of the Serotonin Transporter by the Antidepressant Drugs Trazodone and Nefazodone.

The antidepressants trazodone and nefazodone were approved some 4 and 3 decades ago, respectively. Their action is thought to be mediated, at least in part, by inhibition of the serotonin transporter [SERT/solute carrier (SLC)-6A4]. Surprisingly, their mode of action on SERT has not been characterized. Here, we show that, similar to the chemically related drug vilazodone, trazodone and nefazodone are allosteric ligands: trazodone and nefazodone inhibit uptake by and transport-associated currents through SERT in a mixed-competitive and noncompetitive manner, respectively. Contrary to noribogaine and its congeners, all three compounds preferentially interact with the Na+-bound outward-facing state of SERT. Nevertheless, they act as pharmacochaperones and rescue the folding-deficient variant SERT-P601A/G602A. The vast majority of disease-associated point mutations of SLC6 family members impair folding of the encoded transporter proteins. Our findings indicate that their folding defect can be remedied by targeting allosteric sites on SLC6 transporters. SIGNIFICANCE STATEMENT: The serotonin transporter is a member of the solute carrier-6 family and is the target of numerous antidepressants. Trazodone and nefazodone have long been used as antidepressants. Here, this study shows that their inhibition of the serotonin transporter digressed from the competitive mode seen with other antidepressants. Trazodone and nefazodone rescued a folding-deficient variant of the serotonin transporter. This finding demonstrates that folding defects of mutated solute carrier-6 family members can also be corrected by allosteric ligands.

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.

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.

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.

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.

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.

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.

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.

An Electrophysiological Approach to Measure Changes in the Membrane Surface Potential in Real Time.

Biological membranes carry fixed charges at their surfaces. These arise primarily from phospholipid headgroups. In addition, membrane proteins contribute to the surface potential with their charged residues. Membrane lipids are asymmetrically distributed. Because of this asymmetry, the net-negative charge at the inner leaflet exceeds that at the outer leaflet. Changes in surface potential are predicted to give rise to apparent changes in membrane capacitance. Here, we show that it is possible to detect changes in surface potential by an electrophysiological approach; the analysis of cellular currents relies on assuming that the electrical properties of a cell are faithfully described by a three-element circuit (i.e., the minimal equivalent circuit) comprised of two resistors and one capacitor. However, to account for changes in surface potential, it is necessary to add a battery to this circuit connected in series with the capacitor. This extended circuit model predicts that the current response to a square-wave voltage pulse harbors information, which allows for separating the changes in surface potential from a true capacitance change. We interrogated our model by investigating changes in the capacitance induced by ligand binding to the serotonin transporter and to the glycine transporters (GlyT1 and GlyT2). The experimental observations were consistent with the predictions of the extended circuit. We conclude that ligand-induced changes in surface potential (reflecting the binding event) and in true membrane capacitance (reflecting the concomitant conformational change) can be detected in real time even in instances in which they occur simultaneously.

Functional Impact of the G279S Substitution in the Adenosine A1-Receptor (A1R-G279S7.44), a Mutation Associated with Parkinson's Disease.

In medium-size, spiny striatal neurons of the direct pathway, dopamine D1- and adenosine A1-receptors are coexpressed and are mutually antagonistic. Recently, a mutation in the gene encoding the A1-receptor (A1R), A1R-G279S7.44, was identified in an Iranian family: two affected offspring suffered from early-onset l-DOPA-responsive Parkinson's disease. The link between the mutation and the phenotype is unclear. Here, we explored the functional consequence of the G279S substitution on the activity of the A1-receptor after heterologous expression in HEK293 cells. The mutation did not affect surface expression and ligand binding but changed the susceptibility to heat denaturation: the thermodynamic stability of A1R-G279S7.44 was enhanced by about 2 and 8 K when compared with wild-type A1-receptor and A1R-Y288A7.53 (a folding-deficient variant used as a reference), respectively. In contrast, the kinetic stability was reduced, indicating a lower energy barrier for conformational transitions in A1R-G279S7.44 (73 ± 23 kJ/mol) than in wild-type A1R (135 ± 4 kJ/mol) or in A1R-Y288A7.53 (184 ± 24 kJ/mol). Consistent with this lower energy barrier, A1R-G279S7.44 was more effective in promoting guanine nucleotide exchange than wild-type A1R. We detected similar levels of complexes formed between D1-receptors and wild-type A1R or A1R-G279S7.44 by coimmunoprecipitation and bioluminescence resonance energy transfer. However, lower concentrations of agonist were required for half-maximum inhibition of dopamine-induced cAMP accumulation in cells coexpressing D1-receptor and A1R-G279S7.44 than in those coexpressing wild-type A1R. These observations predict enhanced inhibition of dopaminergic signaling by A1R-G279S7.44 in vivo, consistent with a pathogenic role in Parkinson's disease. SIGNIFICANCE STATEMENT: Parkinson's disease is caused by a loss of dopaminergic input from the substantia nigra to the caudate nucleus and the putamen. Activation of the adenosine A1-receptor antagonizes responses elicited by dopamine D1-receptor. We show that this activity is more pronounced in a mutant version of the A1-receptor (A1R-G279S7.44), which was identified in individuals suffering from early-onset Parkinson's disease.

Effect of rain on absorption after transdermal application of flunixin in calves.

Flunixin is a nonsteroidal anti-inflammatory drug (NSAID) that has anti-inflammatory, anti-pyretic, and analgesic effects. Recently, a novel transdermal formulation was developed (Finadyne® Transdermal, MSD Animal Health) and is now the first NSAID registered to be administered as a pour-on product in cattle. According to the manufacturer's instructions, the pour-on product should be applied only to dry skin and exposure to rain should be avoided for at least 6 hr after application. The objective of the study was to evaluate the effect of simulated exposure to light or heavy rain on flunixin absorption and bioavailability within the first 4 hr after administration. Therefore, an isocratic HPLC method was developed to quantify flunixin concentrations in bovine serum by UV detection. Light rain decreased flunixin absorption only when rain started immediately after flunixin administration, while light rain starting more than 30 min after administration of flunixin had no effect on absorption. Absorption and bioavailability of flunixin was impacted under simulated heavy rain conditions, when exposure to rain occurred within one hour after the application of the pour-on formulation, but not later.

Functional Selectivity and Partial Efficacy at the Monoamine Transporters: A Unified Model of Allosteric Modulation and Amphetamine-Induced Substrate Release.

All clinically approved drugs targeting the plasmalemmal transporters for dopamine, norepinephrine, and serotonin act either as competitive uptake inhibitors or as amphetamine-like releasers. Monoamine transporter (MAT) ligands that allosterically affect MAT-mediated substrate uptake, release, or both were recently discovered. Their modes of action have not yet been explained in a unified framework. Here, we go beyond competitive inhibitors and classic amphetamines and introduce concepts for partial efficacy at and allosteric modulation of MATs. After we elaborate on a kinetic account for amphetamine action, we provide an explanation for partial release (i.e., the observation that some amphetamines are less efficacious than others in inducing monoamine efflux). We then elucidate mechanisms of allosteric inhibition and stimulation of MATs, which can be functionally selective for either substrate uptake or amphetamine-induced release. These concepts are integrated into a parsimonious kinetic framework, which relies exclusively on physiologic transport modes (without any deviation from an alternating access mechanism). The model posits cooperative substrate and Na+ binding and functional selectivity by conformational selection (i.e., preference of the allosteric modulators for the substrate-loaded or substrate-free states of the transporter). Thus, current knowledge about the kinetics of monoamine transport is sufficiently detailed to provide a quantitative description of the releasing action of amphetamines, of substrate uptake, and of selective modulation thereof by allosteric modulators.

How to rescue misfolded SERT, DAT and NET: targeting conformational intermediates with atypical inhibitors and partial releasers.

Point mutations in the coding sequence for solute carrier 6 (SLC6) family members result in clinically relevant disorders, which are often accounted for by a loss-of-function phenotype. In many instances, the mutated transporter is not delivered to the cell surface because it is retained in the endoplasmic reticulum (ER). The underlying defect is improper folding of the transporter and is the case for many of the known dopamine transporter mutants. The monoamine transporters, i.e. the transporters for norepinephrine (NET/SLC6A2), dopamine (DAT/SLC6A3) and serotonin (SERT/SLC6A4), have a rich pharmacology; hence, their folding-deficient mutants lend themselves to explore the concept of pharmacological chaperoning. Pharmacochaperones are small molecules, which bind to folding intermediates with exquisite specificity and scaffold them to a folded state, which is exported from the ER and delivered to the cell surface. Pharmacochaperoning of mutant monoamine transporters, however, is not straightforward: ionic conditions within the ER are not conducive to binding of most typical monoamine transporter ligands. A collection of compounds exists, which are classified as atypical ligands because they trap monoamine transporters in unique conformational states. The atypical binding mode of some DAT inhibitors has been linked to their anti-addictive action. Here, we propose that atypical ligands and also compounds recently classified as partial releasers can serve as pharmacochaperones.

Kinetic Models of Secondary Active Transporters.

Kinetic models have been employed to understand the logic of substrate transport through transporters of the Solute Carrier (SLC) family. All SLC transporters operate according to the alternate access model, which posits that substrate transport occurs in a closed loop of partial reactions (i.e., a transport cycle). Kinetic models can help to find realistic estimates for conformational transitions between individual states of the transport cycle. When constrained by experimental results, kinetic models can faithfully describe the function of a candidate transporter at a pre-steady state. In addition, we show that kinetic models can accurately predict the intra- and extracellular substrate concentrations maintained by the transporter at a steady state, even under the premise of loose coupling between the electrochemical gradient of the driving ion and of the substrate. We define the criteria for the design of a credible kinetic model of the SLC transporter. Parsimony is the guiding principle of kinetic modeling. We argue, however, that the level of acceptable parsimony is limited by the need to account for the substrate gradient established by a secondary active transporter, and for random order binding of co-substrates and substrate. Random order binding has consistently been observed in transporters of the SLC group.

The N Terminus Specifies the Switch between Transport Modes of the Human Serotonin Transporter.

The serotonin transporter (SERT) and other monoamine transporters operate in either a forward transport mode where the transporter undergoes a full transport cycle or an exchange mode where the transporter seesaws through half-cycles. Amphetamines trigger the exchange mode, leading to substrate efflux. This efflux was proposed to rely on the N terminus, which was suggested to adopt different conformations in the inward facing, outward facing and amphetamine-bound states. This prediction was verified by tryptic digestion of SERT-expressing membranes: in the absence of Na+, the N terminus was rapidly digested. Amphetamine conferred protection against cleavage, suggesting a relay between the conformational states of the hydrophobic core and the N terminus. We searched for a candidate segment that supported the conformational switch by serial truncation removing 22 (ΔN22), 32 (ΔN32), or 42 (ΔN42) N-terminal residues. This did not affect surface expression, inhibitor binding, and substrate influx. However, amphetamine-induced efflux by SERT-ΔN32 or SERT-ΔN42 (but not by SERT-ΔN22) was markedly diminished. We examined the individual steps in the transport cycle by recording transporter-associated currents: the recovery rate of capacitive peak, but not of steady state, currents was significantly lower for SERT-ΔN32 than that of wild type SERT and SERT-ΔN22. Thus, the exchange mode of SERT-ΔN32 was selectively impaired. Our observations show that the N terminus affords the switch between transport modes. The findings are consistent with a model where the N terminus acts as a lever to support amphetamine-induced efflux by SERT.

Pharmacochaperoning in a Drosophila model system rescues human dopamine transporter variants associated with infantile/juvenile parkinsonism.

Point mutations in the gene encoding the human dopamine transporter (hDAT, SLC6A3) cause a syndrome of infantile/juvenile dystonia and parkinsonism. To unravel the molecular mechanism underlying these disorders and investigate possible pharmacological therapies, here we examined 13 disease-causing DAT mutants that were retained in the endoplasmic reticulum when heterologously expressed in HEK293 cells. In three of these mutants, i.e. hDAT-V158F, hDAT-G327R, and hDAT-L368Q, the folding deficit was remedied with the pharmacochaperone noribogaine or the heat shock protein 70 (HSP70) inhibitor pifithrin-µ such that endoplasmic reticulum export of and radioligand binding and substrate uptake by these DAT mutants were restored. In Drosophila melanogaster, DAT deficiency results in reduced sleep. We therefore exploited the power of targeted transgene expression of mutant hDAT in Drosophila to explore whether these hDAT mutants could also be pharmacologically rescued in an intact organism. Noribogaine or pifithrin-µ treatment supported hDAT delivery to the presynaptic terminals of dopaminergic neurons and restored sleep to normal length in DAT-deficient (fumin) Drosophila lines expressing hDAT-V158F or hDAT-G327R. In contrast, expression of hDAT-L368Q in the Drosophila DAT mutant background caused developmental lethality, indicating a toxic action not remedied by pharmacochaperoning. Our observations identified those mutations most likely amenable to pharmacological rescue in the affected children. In addition, our findings also highlight the challenges of translating insights from pharmacochaperoning in cell culture to the clinical situation. Because of the evolutionary conservation in dopaminergic neurotransmission between Drosophila and people, pharmacochaperoning of DAT in D. melanogaster may allow us to bridge that gap.

Occupancy of the Zinc-binding Site by Transition Metals Decreases the Substrate Affinity of the Human Dopamine Transporter by an Allosteric Mechanism.

The human dopamine transporter (DAT) has a tetrahedral Zn2+-binding site. Zn2+-binding sites are also recognized by other first-row transition metals. Excessive accumulation of manganese or of copper can lead to parkinsonism because of dopamine deficiency. Accordingly, we examined the effect of Mn2+, Co2+, Ni2+, and Cu2+ on transport-associated currents through DAT and DAT-H193K, a mutant with a disrupted Zn2+-binding site. All transition metals except Mn2+ modulated the transport cycle of wild-type DAT with affinities in the low micromolar range. In this concentration range, they were devoid of any action on DAT-H193K. The active transition metals reduced the affinity of DAT for dopamine. The affinity shift was most pronounced for Cu2+, followed by Ni2+ and Zn2+ (= Co2+). The extent of the affinity shift and the reciprocal effect of substrate on metal affinity accounted for the different modes of action: Ni2+ and Cu2+ uniformly stimulated and inhibited, respectively, the substrate-induced steady-state currents through DAT. In contrast, Zn2+ elicited biphasic effects on transport, i.e. stimulation at 1 µm and inhibition at 10 µm A kinetic model that posited preferential binding of transition metal ions to the outward-facing apo state of DAT and a reciprocal interaction of dopamine and transition metals recapitulated all experimental findings. Allosteric activation of DAT via the Zn2+-binding site may be of interest to restore transport in loss-of-function mutants.

Conformational state interactions provide clues to the pharmacochaperone potential of serotonin transporter partial substrates.

Point mutations in SLC6 transporters cause misfolding, which can be remedied by pharmacochaperones. The serotonin transporter (SERT/SLC6A4) has a rich pharmacology including inhibitors, releasers (amphetamines, which promote the exchange mode), and more recently, discovered partial substrates. We hypothesized that partial substrates trapped the transporter in one or several states of the transport cycle. This conformational trapping may also be conducive to folding. We selected naphthylpropane-2-amines of the phenethylamine library (PAL) including the partial substrate PAL1045 and its congeners PAL287 and PAL1046. We analyzed their impact on the transport cycle of SERT by biochemical approaches and by electrophysiological recordings; substrate-induced peak currents and steady-state currents monitored the translocation of substrate and co-substrate Na+ across the lipid bilayer and the transport cycle, respectively. These experiments showed that PAL1045 and its congeners bound with different affinities (ranging from nm to µm) to various conformational intermediates of SERT during the transport cycle. Consistent with the working hypothesis, PAL1045 was the most efficacious compound in restoring surface expression and transport activity to the folding-deficient mutant SERT-601PG602-AA. These experiments provide a proof-of-principle for a rational search for pharmacochaperones, which may be useful to restore function to clinically relevant folding-deficient transporter mutants.