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

The antidepressants trazodone and nefazodone were approved some four and three decades ago, respectively. Their action is thought to be mediated - at least in part - by inhibition of the serotonin transporter (SERT/SLC6A4). 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, which inhibit uptake by and transport-associated currents through SERT in a mixed-competitive and non-competitive 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 (solute carrier-6) 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 we show that their inhibition of the serotonin transporter digressed from the competiti-ve 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.

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.

Orphan lysosomal solute carrier MFSD1 facilitates highly selective dipeptide transport.

Orphan solute carrier (SLC) represents a group of membrane transporters whose exact functions and substrate specificities are not known. Elucidating the function and regulation of orphan SLC transporters is not only crucial for advancing our knowledge of cellular and molecular biology but can potentially lead to the development of new therapeutic strategies. Here, we provide evidence for the biological function of a ubiquitous orphan lysosomal SLC, the Major Facilitator Superfamily Domain-containing Protein 1 (MFSD1), which has remained phylogenetically unassigned. Targeted metabolomics revealed that dipeptides containing either lysine or arginine residues accumulate in lysosomes of cells lacking MFSD1. Whole-cell patch-clamp electrophysiological recordings of HEK293-cells expressing MFSD1 on the cell surface displayed transport affinities for positively charged dipeptides in the lower mM range, while dipeptides that carry a negative net charge were not transported. This was also true for single amino acids and tripeptides, which MFSD1 failed to transport. Our results identify MFSD1 as a highly selective lysosomal lysine/arginine/histidine-containing dipeptide exporter, which functions as a uniporter.

Structure-based discovery of conformationally selective inhibitors of the serotonin transporter.

The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has unusual anti-depressant and substance-withdrawal effects, and stabilizes the inward-open conformation. Unfortunately, ibogaine's promiscuity and cardiotoxicity limit the understanding of inward-open state ligands. We docked over 200 million small molecules against the inward-open state of the SERT. Thirty-six top-ranking compounds were synthesized, and thirteen inhibited; further structure-based optimization led to the selection of two potent (low nanomolar) inhibitors. These stabilized an outward-closed state of the SERT with little activity against common off-targets. A cryo-EM structure of one of these bound to the SERT confirmed the predicted geometry. In mouse behavioral assays, both compounds had anxiolytic- and anti-depressant-like activity, with potencies up to 200-fold better than fluoxetine (Prozac), and one substantially reversed morphine withdrawal effects.

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.

Trapped Pore Waters in the Open Proton Channel HV 1.

The voltage-gated proton channel, HV 1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, the water mobility for the putative case of an uninterrupted wire is estimated. The predicted single-channel water permeability 2.3 × 10-12 cm3 s-1 reflects the permeability-governing number of hydrogen bonds between water molecules in single-file configuration and pore residues. However, the measured unitary water permeability does not confirm the predicted value. Osmotic deflation of reconstituted lipid vesicles reveals negligible water permeability of the HV 1 wild-type channel and the D174A mutant open at 0 mV. The conductance of 1400 H+ s-1 per wild-type channel agrees with the calculated diffusion limit for a ≈2 Å capture radius for protons. Removal of a charged amino acid (D174) at the pore mouth decreases H+ conductance by reducing the capture radius. At least one intervening amino acid contributes to H+ conductance while interrupting the water wire across the membrane.

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.

Photoswitching of model ion channels in lipid bilayers.

Membrane proteins can be regulated by alterations in material properties intrinsic to the hosting lipid bilayer. Here, we investigated whether the reversible photoisomerization of bilayer-embedded diacylglycerols (OptoDArG) with two azobenzene-containing acyl chains may trigger such regulatory events. We observed an augmented open probability of the mechanosensitive model channel gramicidin A (gA) upon photoisomerizing OptoDArG's acyl chains from trans to cis: integral planar bilayer conductance brought forth by hundreds of simultaneously conducting gA dimers increased by typically >50% - in good agreement with the observed increase in single-channel lifetime. Further, (i) increments in the electrical capacitance of planar lipid bilayers and protrusion length of aspirated giant unilamellar vesicles into suction pipettes, as well as (ii) changes of small-angle X-ray scattering of multilamellar vesicles indicated that spontaneous curvature, hydrophobic thickness, and bending elasticity decreased upon switching from trans- to cis-OptoDArG. Our bilayer elasticity model for gA supports the causal relationship between changes in gA activity and bilayer material properties upon photoisomerization. Thus, we conclude that photolipids are deployable for converting bilayers of potentially diverse origins into light-gated actuators for mechanosensitive proteins.

Nanoscopic Approach to Study the Early Stages of Epithelial to Mesenchymal Transition (EMT) of Human Retinal Pigment Epithelial (RPE) Cells In Vitro.

The maintenance of visual function is supported by the proper functioning of the retinal pigment epithelium (RPE), representing a mosaic of polarized cuboidal postmitotic cells. Damage factors such as inflammation, aging, or injury can initiate the migration and proliferation of RPE cells, whereas they undergo a pseudo-metastatic transformation or an epithelial to mesenchymal transition (EMT) from cuboidal epithelioid into fibroblast-like or macrophage-like cells. This process is recognized as a key feature in several severe ocular pathologies, and is mimicked by placing RPE cells in culture, which provides a reasonable and well-characterized in vitro model for a type 2 EMT. The most obvious characteristic of EMT is the cell phenotype switching, accompanied by the cytoskeletal reorganization with changes in size, shape, and geometry. Atomic force microscopy (AFM) has the salient ability to label-free explore these characteristics. Based on our AFM results supported by the genetic analysis of specific RPE differentiation markers, we elucidate a scheme for gradual transformation from the cobblestone to fibroblast-like phenotype. Structural changes in the actin cytoskeletal reorganization at the early stages of EMT lead to the development of characteristic geodomes, a finding that may reflect an increased propensity of RPE cells to undergo further EMT and thus become of diagnostic significance.

Micropipette Aspiration-Based Assessment of Single Channel Water Permeability.

Measurements of the unitary hydraulic conductivity of membrane channels, pf , may be hampered by difficulties in producing sufficient quantities of purified and reconstituted proteins. Low yield expression, the purely empiric choice of detergents, as well as protein aggregation and misfolding during reconstitution may result in an average of less than one reconstituted channel per large unilamellar vesicle. This limits their applicability for pf measurements, independent of whether light scattering or fluorescence quenching of encapsulated dyes is monitored. Here the micropipette aspiration technique is adopted because its superb sensitivity allows resolving pf values for one order of magnitude smaller protein densities in sphingomyelin and cholesterol rich giant unilamellar vesicles (GUVs). Protein density is derived from intensity fluctuations that fluorescently labeled channels in the aspirated GUV induce by diffusing through the diffraction limited spot. A perfusion system minimizes unstirred layers in the immediate membrane vicinity as demonstrated by the distribution of both encapsulated and extravesicular aqueous dyes. pf amounted to 2.4 ± 0.1 × 10-13 cm³ s-1 for aquaporin-1 that served as a test case. The new assay paves the way for directly monitoring the effect that interaction of aquaporins with other proteins or inhibitors may have on pf on a single sample.

Design of peptide-membrane interactions to modulate single-file water transport through modified gramicidin channels.

Water permeability through single-file channels is affected by intrinsic factors such as their size and polarity and by external determinants like their lipid environment in the membrane. Previous computational studies revealed that the obstruction of the channel by lipid headgroups can be long-lived, in the range of nanoseconds, and that pore-length-matching membrane mimetics could speed up water permeability. To test the hypothesis of lipid-channel interactions modulating channel permeability, we designed different gramicidin A derivatives with attached acyl chains. By combining extensive molecular-dynamics simulations and single-channel water permeation measurements, we show that by tuning lipid-channel interactions, these modifications reduce the presence of lipid headgroups in the pore, which leads to a clear and selective increase in their water permeability.