A previously unrecognized superfamily of macro-conotoxins includes an inhibitor of the sensory neuron calcium channel Cav2.3.

Animal venom peptides represent valuable compounds for biomedical exploration. The venoms of marine cone snails constitute a particularly rich source of peptide toxins, known as conotoxins. Here, we identify the sequence of an unusually large conotoxin, Mu8.1, which defines a new class of conotoxins evolutionarily related to the well-known con-ikot-ikots and 2 additional conotoxin classes not previously described. The crystal structure of recombinant Mu8.1 displays a saposin-like fold and shows structural similarity with con-ikot-ikot. Functional studies demonstrate that Mu8.1 curtails calcium influx in defined classes of murine somatosensory dorsal root ganglion (DRG) neurons. When tested on a variety of recombinantly expressed voltage-gated ion channels, Mu8.1 displayed the highest potency against the R-type (Cav2.3) calcium channel. Ca2+ signals from Mu8.1-sensitive DRG neurons were also inhibited by SNX-482, a known spider peptide modulator of Cav2.3 and voltage-gated K+ (Kv4) channels. Our findings highlight the potential of Mu8.1 as a molecular tool to identify and study neuronal subclasses expressing Cav2.3. Importantly, this multidisciplinary study showcases the potential of uncovering novel structures and bioactivities within the largely unexplored group of macro-conotoxins.

Identification and functional evaluation of GRIA1 missense and truncation variants in individuals with ID: An emerging neurodevelopmental syndrome.

GRIA1 encodes the GluA1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors, which are ligand-gated ion channels that act as excitatory receptors for the neurotransmitter L-glutamate (Glu). AMPA receptors (AMPARs) are homo- or heteromeric protein complexes with four subunits, each encoded by different genes, GRIA1 to GRIA4. Although GluA1-containing AMPARs have a crucial role in brain function, the human phenotype associated with deleterious GRIA1 sequence variants has not been established. Subjects with de novo missense and nonsense GRIA1 variants were identified through international collaboration. Detailed phenotypic and genetic assessments of the subjects were carried out and the pathogenicity of the variants was evaluated in vitro to characterize changes in AMPAR function and expression. In addition, two Xenopus gria1 CRISPR-Cas9 F0 models were established to characterize the in vivo consequences. Seven unrelated individuals with rare GRIA1 variants were identified. One individual carried a homozygous nonsense variant (p.Arg377Ter), and six had heterozygous missense variations (p.Arg345Gln, p.Ala636Thr, p.Ile627Thr, and p.Gly745Asp), of which the p.Ala636Thr variant was recurrent in three individuals. The cohort revealed subjects to have a recurrent neurodevelopmental disorder mostly affecting cognition and speech. Functional evaluation of major GluA1-containing AMPAR subtypes carrying the GRIA1 variant mutations showed that three of the four missense variants profoundly perturb receptor function. The homozygous stop-gain variant completely destroys the expression of GluA1-containing AMPARs. The Xenopus gria1 models show transient motor deficits, an intermittent seizure phenotype, and a significant impairment to working memory in mutants. These data support a developmental disorder caused by both heterozygous and homozygous variants in GRIA1 affecting AMPAR function.

Three-dimensional missense tolerance ratio analysis.

A wealth of genetic information is available describing single-nucleotide variants in the human population that appear to be well-tolerated and in and of themselves do not confer disease. These variant data sets contain signatures about the protein structure-function relationships and provide an unbiased view of various protein functions in the context of human health. This information can be used to determine regional intolerance to variation, defined as the missense tolerance ratio (MTR), which is an indicator of stretches of the polypeptide chain that can tolerate changes without compromising protein function in a manner that impacts human health. This approach circumvents the lack of comprehensive data by averaging the data from adjacent residues on the polypeptide chain. We reasoned that many motifs in proteins consist of nonadjacent residues, but together function as a unit. We therefore developed an approach to analyze nearest neighbors in three-dimensional space as determined by crystallography rather than on the polypeptide chain. We used members of the GRIN gene family that encode subunits of NMDA-type ionotropic glutamate receptors (iGluRs) to exemplify the differences between these methods. Our method, 3DMTR, provides new information about regions of intolerance within iGluRs, allows consideration of protein-protein interfaces in multimeric proteins, and moves this important research tool from one-dimensional analysis to a structurally relevant tool. We validate the improved 3DMTR score by showing that it more accurately classifies the functional consequences of a set of newly measured and published point mutations of Grin family genes than existing methods.

Discovery of the First Highly Selective Antagonist of the GluK3 Kainate Receptor Subtype.

Kainate receptors belong to the family of glutamate receptors ion channels, which are responsible for the majority of rapid excitatory synaptic transmission in the central nervous system. The therapeutic potential of kainate receptors is still poorly understood, which is also due to the lack of potent and subunit-selective pharmacological tools. In search of selective ligands for the GluK3 kainate receptor subtype, a series of quinoxaline-2,3-dione analogues was synthesized and pharmacologically characterized at selected recombinant ionotropic glutamate receptors. Among them, compound 28 was found to be a competitive GluK3 antagonist with submicromolar affinity and unprecedented high binding selectivity, showing a 400-fold preference for GluK3 over other homomeric receptors GluK1, GluK2, GluK5 and GluA2. Furthermore, in functional assays performed for selected metabotropic glutamate receptor subtypes, 28 did not show agonist or antagonist activity. The molecular determinants underlying the observed affinity profile of 28 were analyzed using molecular docking and molecular dynamics simulations performed for individual GluK1 and GluK3 ligand-binding domains.

Conformational Changes in the 5-HT3A Receptor Extracellular Domain Measured by Voltage-Clamp Fluorometry.

The 5-hydroxytryptamine (5-HT) type 3 receptor is a member of the cysteine (Cys)-loop receptor super family of ligand-gated ion channels in the nervous system and is a clinical target in a range of diseases. The 5-HT3 receptor mediates fast serotonergic neurotransmission by undergoing a series of conformational changes initiated by ligand binding that lead to the rapid opening of an intrinsic cation-selective channel. However, despite the availability of high-resolution structures of a mouse 5-HT3 receptor, many important aspects of the mechanistic basis of 5-HT3 receptor function and modulation by drugs remain poorly understood. In particular, there is little direct evidence for the specific conformational changes predicted to occur during ligand-gated channel activation and desensitization. In the present study, we used voltage-clamp fluorometry (VCF) to measure conformational changes in regions surrounding the orthosteric binding site of the human 5-HT3A (h5-HT3A) receptor during binding of 5-HT and different classes of 5-HT3 receptor ligands. VCF utilizes parallel measurements of receptor currents with photon emission from fluorescent reporter groups covalently attached to specific positions in the receptor structure. Reporter groups that are highly sensitive to the local molecular environment can, in real time, report conformational changes as changes in fluorescence that can be correlated with changes in receptor currents reporting the functional states of the channel. Within the loop C, D, and E regions that surround the orthosteric binding site in the h5-HT3A receptor, we identify positions that are amenable to tagging with an environmentally sensitive reporter group that reports robust fluorescence changes upon 5-HT binding and receptor activation. We use these reporter positions to characterize the effect of ligand binding on the local structure of the orthosteric binding site by agonists, competitive antagonists, and allosterically acting channel activators. We observed that loop C appears to show distinct fluorescence changes for ligands of the same class, while loop D reports similar fluorescence changes for all ligands binding at the orthosteric site. In contrast, the loop E reporter position shows distinct changes for agonists, antagonists, and allosteric compounds, suggesting the conformational changes in this region are specific to ligand function. Interpretation of these results within the framework of current models of 5-HT3 and Cys-loop mechanisms are used to expand the understanding of how ligand binding in Cys-loop receptors relates to channel gating. SIGNIFICANCE STATEMENT: The 5-HT3 receptor is an important ligand-gated ion channel and drug target in the central and peripheral nervous system. Determining how ligand binding induced conformational changes in the receptor is central for understanding the structural mechanisms underlying 5-HT3 receptor function. Here, we employ voltage-gated fluorometry to characterize conformational changes in the extracellular domain of the human 5-HT3 receptor to identify intrareceptor motions during binding of a range of 5-HT3 receptor agonists and antagonists.

Mutational Analysis and Modeling of Negative Allosteric Modulator Binding Sites in AMPA Receptors.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) constitute a subclass of the ionotropic glutamate receptor superfamily, which functions as glutamate-gated cation channels to mediate the majority of excitatory neurotransmission in the central nervous system. AMPARs are therapeutic targets in a range of brain disorders associated with abnormal glutamate hyperactivity. Multiple classes of AMPAR inhibitors have been developed during the past decades, including competitive antagonists, ion channel blockers, and negative allosteric modulators (NAMs). At present, the NAM is the only class of AMPAR ligands that have been developed into safe and useful drugs in humans in the form of perampanel (Fycompa), which was recently approved for treatment of epilepsy. Compared with the detailed understanding of other AMPAR ligand classes, surprisingly little information has been available regarding the molecular mechanism of perampanel and other classes of NAMs at AMPARs; including the location and structure of NAM binding pockets in the receptor complex. However, structures of the AMPAR GluA2 in complex with NAMs were recently reported that unambiguously identified the NAM binding sites. In parallel with this work, our aim with the present study was to identify specific residues involved in the formation of the NAM binding site for three prototypical AMPAR NAMs. Hence, we have performed a mutational analysis of the AMPAR region that links the four extracellular ligand-binding domains to the central ion channel in the transmembrane domain region. Furthermore, we perform computational ligand docking of the NAMs into structural models of the homomeric GluA2 receptor and optimize side chain conformations around the NAMs to model how NAMs bind in this specific site. The new insights provide potentially valuable input for structure-based drug design of new NAMs. SIGNIFICANCE STATEMENT: The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are glutamate-gated ion channels that mediate the majority of excitatory neurotransmission in the brain. Negative allosteric modulators of AMPA receptors are considered to have significant therapeutic potential in diseases linked to glutamate hyperactivity. The present work employs mutational analysis and molecular modeling of the binding site for prototypical NAMs to provide new molecular insight into how NAMs interact with the AMPA receptor, which is of potential use for future design of new types of NAMs.

Structural rearrangement of the intracellular domains during AMPA receptor activation.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact.

Modeling and Mutational Analysis of the Binding Mode for the Multimodal Antidepressant Drug Vortioxetine to the Human 5-HT3A Receptor.

5-Hydroxytryptamine3 (5-HT3) receptors are ligand-gated ion channels that mediate neurotransmission by serotonin in the central nervous system. Pharmacological inhibition of 5-HT3 receptor activity has therapeutic potential in several psychiatric diseases, including depression and anxiety. The recently approved multimodal antidepressant vortioxetine has potent inhibitory activity at 5-HT3 receptors. Vortioxetine has an inhibitory mechanism that differs from classic 5-HT3 receptor competitive antagonists despite being believed to bind in the same binding site. Specifically, vortioxetine shows partial agonist activity followed by persistent and insurmountable inhibition. We have investigated the binding mode of vortioxetine at the human 5-HT3A receptor through computational and in vitro experiments to provide insight into the molecular mechanisms behind the unique pharmacological profile of the drug. We find that vortioxetine binds in a manner different from currently known 5-HT3A orthosteric ligands. Specifically, while the binding pattern of vortioxetine mimics some aspects of both the setron class of competitive antagonists and 5-hydroxytryptamine (5-HT) with regards to interactions with residues of the aromatic box motif in the orthosteric binding site, vortioxetine also forms interactions with residues not previously described to be important for the binding of either setrons or 5-HT such as Val202 on Loop F. Our results expand the framework for understanding how orthosteric ligands drive 5-HT3 receptor function, which is of importance for the potential future development of novel classes of 5-HT3 receptor antagonists.

Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors.

The GluD2 receptor is a fundamental component of postsynaptic sites in Purkinje neurons, and is required for normal cerebellar function. GluD2 and the closely related GluD1 are classified as members of the ionotropic glutamate receptor (iGluR) superfamily on the basis of sequence similarity, but do not bind l-glutamate. The amino acid neurotransmitter D-Ser is a GluD2 receptor ligand, and endogenous D-Ser signaling through GluD2 has recently been shown to regulate endocytosis of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type iGluRs during synaptic plasticity in the cerebellum, such as long-term depression. Here, we investigate the pharmacology of the orthosteric binding site in GluD2 by examining the activity of analogs of D-Ser and GluN1 glycine site competitive antagonists at GluD2 receptors containing the lurcher mutation (GluD2(LC)), which promotes spontaneous channel activation. We identify several compounds that modulate GluD2(LC), including a halogenated alanine analog as well as the kynurenic acid analog 7-chloro-4-oxo-1H-quinoline-2-carboxylic acid (7-chlorokynurenic acid; 7-CKA). By correlating thermodynamic and structural data for 7-CKA binding to the isolated GluD2 ligand binding domain (GluD2-LBD), we find that binding 7-CKA to GluD2-LBD differs from D-Ser by inducing an intermediate cleft closure of the clamshell-shaped LBD. The GluD2 ligands identified here can potentially serve as a starting point for development of GluD2-selective ligands useful as tools in studies of the signaling role of the GluD2 receptor in the brain.

Substrate and Inhibitor-Specific Conformational Changes in the Human Serotonin Transporter Revealed by Voltage-Clamp Fluorometry.

The serotonin transporter (SERT) regulates neurotransmission by the biogenic monoamine neurotransmitter serotonin (5-HT, 5-hydroxytryptamine) in the central nervous system, and drugs inhibiting SERT are widely used for the treatment of a variety of central nervous system diseases. The conformational dynamics of SERT transport function and inhibition is currently poorly understood. We used voltage-clamp fluorometry to study conformational changes in human SERT (hSERT) during 5-HT transport and inhibitor binding. Cys residues were introduced at 12 positions in hSERT to enable covalent attachment of a rhodamine-based fluorophore. Transport-associated changes in fluorescence from fluorophore-labeled hSERT expressed in Xenopus oocytes could be robustly detected at four positions in hSERT: endogenous Cys109 in the top of transmembrane domain (TM) 1b, Cys substituted for Thr323 in the top of TM6, Ala419 in the interface between TM8 and extracellular loop (EL) 4, and Leu481 in EL5. The reporter positions were used for time-resolved measurement of conformational changes during 5-HT transport and binding of cocaine and the selective serotonin reuptake inhibitors fluoxetine and escitalopram. At all reporter positions, fluorescence changes observed upon substrate application were distinctly different from those observed upon inhibitor application, with respect to relative amplitude or direction. Furthermore, escitalopram, fluoxetine, and cocaine induced a very similar pattern of fluorescent changes overall, which included movements within or around TM1b, EL4, and EL5. Taken together, our data lead us to suggest that competitive inhibitors stabilize hSERT in a state that is different from the apo outward-open conformation as well as inward-facing conformations.

A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage.

Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-D-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4(IETDV)(2) (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.

Evaluation of PhTX-74 as subtype-selective inhibitor of GluA2-containing AMPA receptors.

The α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are glutamate-gated cation channels that mediate fast excitatory synaptic transmission in the central nervous system. AMPARs are tetramers formed by homo- or heteromeric assembly of GluA1-4 subunits to produce multiple subtypes with varying biophysical properties. Polyamine toxins such as joro spider toxins, philanthotoxins (PhTXs), and argiotoxins are use-dependent ion channel blockers of AMPARs widely employed as highly potent antagonists of GluA2-lacking receptor subtypes. In addition to this use, recent findings have indicated that a philanthotoxin analog, PhTX-74, can distinguish among GluA2-containing AMPAR subtypes in the presence of the prototypical transmembrane AMPAR regulatory protein γ-2 (or stargazin). Thus, PhTX-74 may be of potential use in studies of the neurobiological role of GluA2-containing subtypes. We have evaluated the pharmacological profile of PhTX-74 and related polyamine toxins at homo- and heteromeric AMPARs in the presence and absence of γ-2. Determination of IC(50) values for inhibition of glutamate-evoked currents from Xenopus oocytes expressing recombinant homo- or heteromeric combinations of GluA1, GluA2, and GluA3 in the presence of γ-2 shows that PhTX-74 inhibits homomeric GluA1 and GluA3 receptors nonselectively, with IC(50) values in the nanomolar range (252-356 nM), and heteromeric GluA1/A2 and GluA2/A3 receptors nonselectively, with IC(50) values in the micromolar range (22 µM). Thus, in contrast to earlier findings, we find that PhTX-74 cannot pharmacologically discriminate between GluA2-containing AMPAR subtypes.

Molecular basis for selective serotonin reuptake inhibition by the antidepressant agent fluoxetine (Prozac).

Inhibitors of the serotonin transporter (SERT) are widely used antidepressant agents, but the structural mechanism for inhibitory activity and selectivity over the closely related norepinephrine transporter (NET) is not well understood. Here we use a combination of chemical, biological, and computational methods to decipher the molecular basis for high-affinity recognition in SERT and selectivity over NET for the prototypical antidepressant drug fluoxetine (Prozac; Eli Lilly, Indianapolis, IN). We show that fluoxetine binds within the central substrate site of human SERT, in agreement with recent X-ray crystal structures of LeuBAT, an engineered monoamine-like version of the bacterial amino acid transporter LeuT. However, the binding orientation of fluoxetine is reversed in our experimentally supported model compared with the LeuBAT structures, emphasizing the need for careful experimental verification when extrapolating findings from crystal structures of bacterial transporters to human relatives. We find that the selectivity of fluoxetine and nisoxetine, a NET selective structural congener of fluoxetine, is controlled by residues in different regions of the transporters, indicating a complex mechanism for selective recognition of structurally similar compounds in SERT and NET. Our findings add important new information on the molecular basis for SERT/NET selectivity of antidepressants, and provide the first assessment of the potential of LeuBAT as a model system for antidepressant binding in human transporters, which is essential for future structure-based drug development of antidepressant drugs with fine-tuned transporter selectivity.

Inhibition of AMPA receptors by polyamine toxins is regulated by agonist efficacy and stargazin.

The α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are glutamate-gated cation channels mediating the majority of fast excitatory synaptic transmission in the central nervous system (CNS). Polyamine toxins derived from spiders and wasps are use- and voltage-dependent channel blockers of Ca(2+)-permeable AMPARs. Recent studies have suggested that AMPAR block by polyamine toxins is modulated by auxiliary subunits from the class of transmembrane AMPAR regulatory proteins (TARPs), which may have implications for their use as tool compounds in native systems. We have explored the effect of the TARP γ-2 (also known as stargazin) on the inhibitory potency of three structurally different polyamine toxins at Ca(2+)-permeable homomeric GluA1 AMPARs expressed in oocytes. We find that polyamine toxin IC50 is differentially affected by presence of stargazin depending on the efficacy of the agonists used to activate GluA1. Co-assembly of GluA1 receptors with stargazin increases the potency of the polyamine toxins when activated by the weak partial agonist kainate, but has no effect in presence of full-agonist L-glutamate (Glu) and partial agonist (RS)-willardiine.

Analogues of 3-hydroxyisoxazole-containing glutamate receptor ligands based on the 3-hydroxypyrazole-moiety: design, synthesis and pharmacological characterization.

A series of analogues of the glutamate receptor ligands (S)-2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propionic acid (AMPA) and AMOA were synthesized in which the 3-hydroxyisoxazole moiety was exchanged for a 3-hydroxypyrazole moiety. This exchange enables further substitution at the additional nitrogen atom in the heterocyclic core. Several of the analogues have activity at AMPA receptors equipotent to the antagonist ATPO, demonstrating that additional substitution can be accommodated in the antagonist binding site. Modelling studies offer an explanation for the pharmacological pattern observed for the compounds and suggest that this scaffold may be developed further to obtain subtype selective antagonists.

Molecular determinants for selective recognition of antidepressants in the human serotonin and norepinephrine transporters.

Inhibitors of the serotonin transporter (SERT) and norepinephrine transporter (NET) are widely used in the treatment of major depressive disorder. Although SERT/NET selectivity is a key determinant for the therapeutic properties of these drugs, the molecular determinants defining SERT/NET selectivity are poorly understood. In this study, the structural basis for selectivity of the SERT selective inhibitor citalopram and the structurally closely related NET selective inhibitor talopram is delineated. A systematic structure-activity relationship study allowed identification of the substituents that control activity and selectivity toward SERT and NET and revealed a common pattern showing that SERT and NET have opposite preference for the stereochemical configuration of these inhibitors. Mutational analysis of nonconserved SERT/NET residues within the central substrate binding site was performed to determine the molecular basis for inhibitor selectivity. Changing only five residues in NET to the complementary residues in SERT transferred a SERT-like affinity profile for R- and S-citalopram into NET, showing that the selectivity of these compounds is determined by amino acid differences in the central binding site of the transporters. In contrast, the activity of R- and S-talopram was largely unaffected by any mutations within the central substrate binding site of SERT and NET and in the outer vestibule of NET, suggesting that citalopram and talopram bind to distinct sites on SERT and NET. Together, these findings provide important insight into the molecular basis for SERT/NET selectivity of antidepressants, which can be used to guide rational development of unique transporter inhibitors with fine-tuned transporter selectivity.

SLC6 neurotransmitter transporters: structure, function, and regulation.

The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na(+)/Cl(-)-dependent transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane transporters for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.

Small-molecule positive allosteric modulation of homomeric kainate receptors GluK1-3: development of screening assays and insight into GluK3 structure.

The kainate receptors GluK1-3 (glutamate receptor ionotropic, kainate receptors 1-3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain, and are associated with neurological and psychiatric diseases. How these receptors can be modulated by small-molecule agents is not well understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium-sensitive fluorescence-based assays to test agonists, antagonists, and positive allosteric modulators of GluK1-3. The half-maximal effective concentration (EC50) of BPAM344 for potentiating the response of 100 µm kainate was determined to be 26.3 µm for GluK1, 75.4 µm for GluK2, and 639 µm for GluK3. Domoate was found to be a potent agonist for GluK1 and GluK2, with an EC50 of 0.77 and 1.33 µm, respectively, upon co-application of 150 µm BPAM344. At GluK3, domoate acts as a very weak agonist or antagonist with a half-maximal inhibitory concentration (IC50) of 14.5 µm, in presence of 500 µm BPAM344 and 100 µm kainate for competition binding. Using H523A-mutated GluK3, we determined the first dimeric structure of the ligand-binding domain by X-ray crystallography, allowing location of BPAM344, as well as zinc-, sodium-, and chloride-ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790, and Glu797 in the binding of zinc ions. Using electron microscopy, we show that, in presence of glutamate and BPAM344, full-length GluK3 adopts a dimer-of-dimers arrangement.

Structural determinants for activity of the antidepressant vortioxetine at human and rodent 5-HT3 receptors.

Vortioxetine (VTX) is a recently approved antidepressant that targets a variety of serotonin receptors. Here, we investigate the drug's molecular mechanism of operation at the serotonin 5-HT3 receptor (5-HT3R), which features two properties: VTX acts differently on rodent and human 5-HT3R, and VTX appears to suppress any subsequent response to agonists. Using a combination of cryo-EM, electrophysiology, voltage-clamp fluorometry and molecular dynamics, we show that VTX stabilizes a resting inhibited state of the mouse 5-HT3R and an agonist-bound-like state of human 5-HT3R, in line with the functional profile of the drug. We report four human 5-HT3R structures and show that the human receptor transmembrane domain is intrinsically fragile. We also explain the lack of recovery after VTX administration via a membrane partition mechanism.

Interaction of antidepressants with the serotonin and norepinephrine transporters: mutational studies of the S1 substrate binding pocket.

The serotonin transporter (SERT) and the norepinephrine transporter (NET) are sodium-dependent neurotransmitter transporters responsible for reuptake of released serotonin and norepinephrine, respectively, into nerve terminals in the brain. A wide range of inhibitors of SERT and NET are used as treatment of depression and anxiety disorders or as psychostimulant drugs of abuse. Despite their clinical importance, the molecular mechanisms by which various types of antidepressant drugs bind and inhibit SERT and NET are still elusive for the majority of the inhibitors, including the molecular basis for SERT/NET selectivity. Mutational analyses have suggested that a central substrate binding site (denoted the S1 pocket) also harbors an inhibitor binding site. In this study, we determine the effect of mutating six key S1 residues in human SERT (hSERT) and NET (hNET) on the potency of 15 prototypical SERT/NET inhibitors belonging to different drug classes. Analysis of the resulting drug sensitivity profiles provides novel information on drug binding modes in hSERT and hNET and identifies specific S1 residues as important molecular determinants for inhibitor potency and hSERT/hNET selectivity.