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.

The Binding of Palonosetron and Other Antiemetic Drugs to the Serotonin 5-HT3 Receptor.

Inaccurately perceived as niche drugs, antiemetics are key elements of cancer treatment alleviating the most dreaded side effect of chemotherapy. Serotonin 5-HT3 receptor antagonists are the most commonly prescribed class of drugs to control chemotherapy-induced nausea and vomiting. These antagonists have been clinically successful drugs since the 1980s, yet our understanding of how they operate at the molecular level has been hampered by the difficulty of obtaining structures of drug-receptor complexes. Here, we report the cryoelectron microscopy structure of the palonosetron-bound 5-HT3 receptor. We investigate the binding of palonosetron, granisetron, dolasetron, ondansetron, and cilansetron using molecular dynamics, covering the whole set of antagonists used in clinical practice. The structural and computational results yield detailed atomic insight into the binding modes of the drugs. In light of our data, we establish a comprehensive framework underlying the inhibition mechanism by the -setron drug family.

Conformational transitions of the serotonin 5-HT3 receptor.

The serotonin 5-HT3 receptor is a pentameric ligand-gated ion channel (pLGIC). It belongs to a large family of receptors that function as allosteric signal transducers across the plasma membrane1,2; upon binding of neurotransmitter molecules to extracellular sites, the receptors undergo complex conformational transitions that result in transient opening of a pore permeable to ions. 5-HT3 receptors are therapeutic targets for emesis and nausea, irritable bowel syndrome and depression3. In spite of several reported pLGIC structures4-8, no clear unifying view has emerged on the conformational transitions involved in channel gating. Here we report four cryo-electron microscopy structures of the full-length mouse 5-HT3 receptor in complex with the anti-emetic drug tropisetron, with serotonin, and with serotonin and a positive allosteric modulator, at resolutions ranging from 3.2 Å to 4.5 Å. The tropisetron-bound structure resembles those obtained with an inhibitory nanobody5 or without ligand9. The other structures include an 'open' state and two ligand-bound states. We present computational insights into the dynamics of the structures, their pore hydration and free-energy profiles, and characterize movements at the gate level and cation accessibility in the pore. Together, these data deepen our understanding of the gating mechanism of pLGICs and capture ligand binding in unprecedented detail.

Zinc alleviates pain through high-affinity binding to the NMDA receptor NR2A subunit.

Zinc is abundant in the central nervous system and regulates pain, but the underlying mechanisms are unknown. In vitro studies have shown that extracellular zinc modulates a plethora of signaling membrane proteins, including NMDA receptors containing the NR2A subunit, which display exquisite zinc sensitivity. We created NR2A-H128S knock-in mice to investigate whether Zn2+-NR2A interaction influences pain control. In these mice, high-affinity (nanomolar) zinc inhibition of NMDA currents was lost in the hippocampus and spinal cord. Knock-in mice showed hypersensitivity to radiant heat and capsaicin, and developed enhanced allodynia in inflammatory and neuropathic pain models. Furthermore, zinc-induced analgesia was completely abolished under both acute and chronic pain conditions. Our data establish that zinc is an endogenous modulator of excitatory neurotransmission in vivo and identify a new mechanism in pain processing that relies on NR2A NMDA receptors. The study also potentially provides a molecular basis for the pain-relieving effects of dietary zinc supplementation.

Structural basis of NR2B-selective antagonist recognition by N-methyl-D-aspartate receptors.

N-Methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors endowed with unique pharmacological and functional properties. In particular, their high permeability to calcium ions confers on NMDARs a central role in triggering long term changes in synaptic strength. Under excitotoxic pathological conditions, such as those occurring during brain trauma, stroke, or Parkinson's or Huntington's diseases, calcium influx through NMDAR channels can also lead to neuronal injury. This argues for the use of NMDAR antagonists as potential therapeutic agents. To date, the most promising NMDAR antagonists are ifenprodil and derivatives, compounds that act as noncompetitive inhibitors selective for NMDARs containing the NR2B subunit. Recent studies have identified the large N-terminal domain (NTD) of NR2B as the region controlling ifenprodil sensitivity of NMDARs. We present here a detailed characterization of the ifenprodil binding site using both experimental and computational approaches. 3D homology modeling reveals that ifenprodil fits well in a closed cleft conformation of the NRB NTD; however, ifenprodil can adopt either of two possible binding orientations of opposite direction. By studying the effects of cleft mutations, we show that only the orientation in which the phenyl moiety points deep toward the NTD hinge is functionally relevant. Moreover, based on our model, we identify novel NTD NR2B residues that are crucial for conferring ifenprodil sensitivity and provide functional evidence that these residues directly interact with the ifenprodil molecule. This work provides a general insight into the origin of the subunit-selectivity of NMDAR noncompetitive antagonists and offer clues for the discovery of novel NR2B-selective antagonists.

Reactive derivatives for affinity labeling in the ifenprodil site of NMDA receptors.

To prepare thiol-reactive ifenprodil derivatives designed as potential probes for cysteine-substituted NR2B containing NMDA receptors, electrophilic centers were introduced in different areas of the ifenprodil structure. Intermediates and final compounds were evaluated by binding studies and by electrophysiology to determine the structural requirements for their selectivity. The reactive compounds were further tested for their stability and for their reactivity in model reactions; some were found suitable as structural probes to investigate the binding site and the docking mode of ifenprodil in the NR2B subunit.

Structural rearrangements of NR1/NR2A NMDA receptors during allosteric inhibition.

Ionotropic glutamate receptor (iGluR) subunits contain a large N-terminal domain (NTD) that precedes the agonist-binding domain (ABD) and participates in subunit oligomerization. In NMDA receptors (NMDARs), the NTDs of NR2A and NR2B subunits also form binding sites for the endogenous inhibitor Zn(2+) ion. Although these allosteric sites have been characterized in detail, the molecular mechanisms by which the NTDs communicate with the rest of the receptor to promote its inhibition remain unknown. Here, we identify the ABD dimer interface as a major structural determinant that permits coupling between the NTDs and the channel gate. The strength of this interface also controls proton inhibition, another form of allosteric modulation of NMDARs. Conformational rearrangements at the ABD dimer interface thus appear to be a key mechanism conserved in all iGluR subfamilies, but have evolved to fulfill different functions: fast desensitization at AMPA and kainate receptors, allosteric inhibition at NMDARs.

Synthesis and biological evaluation of 1-amino-2-phosphonomethylcyclopropanecarboxylic acids, new group III metabotropic glutamate receptor agonists.

Stereoisomers of 1-amino-2-phosphonomethylcyclopropanecarboxylic acid (APCPr), conformationally restricted analogues of L-AP4 (2-amino-4-phosphonobutyric acid), have been prepared and evaluated at recombinant group III metabotropic glutamate receptors. They activate these receptors over a broad range of potencies. The most potent isomer (1S,2R)-APCPr displays a similar pharmacological profile as that of L-AP4 (EC50 0.72, 1.95, >500, 0.34 microM at mGlu4, 6, 7, 8 receptors, respectively, and no effect at group I/II mGluRs). It was characterized on native receptors located in the basal ganglia (BG) where it induced a robust and reversible inhibition of synaptic transmission. It was tested in vivo in haloperidol-induced catalepsy, a model of Parkinsonian akinesia, by direct infusion in the globus pallidus of the BG. At a dose of 0.5 nmol/microL, catalepsy was significantly antagonized. This study reveals that (1S,2R)-APCPr is a potent group III mGluR agonist and confirms that these receptors may be considered as a therapeutic target in the Parkinson's disease.

NMDA receptor subunits: function and pharmacology.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels widely expressed in the central nervous system that play key roles in excitatory synaptic transmission. Because of their involvement in numerous neurological disorders, NMDARs are also targets of therapeutic interest. NMDARs occur as multiple subtypes which differ in their subunit composition and in their biophysical and pharmacological properties. In particular, NMDARs contain a diversity of sites at which endogenous ligands or pharmacological agents can act to modulate receptor activity in a subunit-selective manner, and recent structural and functional data have started to reveal the molecular determinants for this subunit selectivity. These include the binding sites for glutamate, the ion-channel pore and the recently identified allosteric sites on the N-terminal domain. Other potential sites yet unexplored by medicinal chemistry programs are also considered, in particular at the interface between subunits. Given the growing body of evidence that diverse brain disorders implicate different NMDAR subtypes, such as NR2B in pain or NR3A in white matter injury, there is a growing interest in exploiting the pharmacological heterogeneity of NMDARs for the development of novel NMDAR subtype-selective compounds.

A prokaryotic proton-gated ion channel from the nicotinic acetylcholine receptor family.

Ligand-gated ion channels (LGICs) mediate excitatory and inhibitory transmission in the nervous system. Among them, the pentameric or 'Cys-loop' receptors (pLGICs) compose a family that until recently was found in only eukaryotes. Yet a recent genome search identified putative homologues of these proteins in several bacterial species. Here we report the cloning, expression and functional identification of one of these putative homologues from the cyanobacterium Gloeobacter violaceus. It was expressed as a homo-oligomer in HEK 293 cells and Xenopus oocytes, generating a transmembrane cationic channel that is opened by extracellular protons and shows slow kinetics of activation, no desensitization and a single channel conductance of 8 pS. Electron microscopy and cross-linking experiments of the protein fused to the maltose-binding protein and expressed in Escherichia coli are consistent with a homo-pentameric organization. Sequence comparison shows that it possesses a compact structure, with the absence of the amino-terminal helix, the canonical disulphide bridge and the large cytoplasmic domain found in eukaryotic pLGICs. Therefore it embodies a minimal structure required for signal transduction. These data establish the prokaryotic origin of the family. Because Gloeobacter violaceus carries out photosynthesis and proton transport at the cytoplasmic membrane, this new proton-gated ion channel might contribute to adaptation to pH change.

A startling role for synaptic zinc.

It has long been known that the synaptic vesicles of certain glutamatergic terminals, as well as some inhibitory terminals, are richly supplied with zinc ions, yet the functional role of this pool of zinc in synaptic transmission has remained elusive. In this issue of Neuron, Hirzel et al. provide direct in vivo evidence that endogenous zinc is required for proper functioning of neuronal circuitry in the brainstem and spinal cord. They show that knockin mice carrying a point mutation which eliminates zinc potentiation of alpha1-containing glycine receptors develop severe sensorimotor deficits characteristic of impaired glycinergic neurotransmission.

The micromolar zinc-binding domain on the NMDA receptor subunit NR2B.

Eukaryotic ionotropic glutamate receptor subunits possess a large N-terminal domain (NTD) distinct from the neighboring agonist-binding domain. In NMDA receptors, the NTDs of NR2A and NR2B form modulatory domains binding allosteric inhibitors. Despite a high sequence homology, these two domains have been shown to bind two ligands of strikingly different chemical nature. Whereas the NTD of NR2A binds zinc with high (nanomolar) affinity, the NTD of NR2B binds the synthetic neuroprotectant ifenprodil and its derivatives. Using both NTD-mutated/deleted receptors and isolated NTDs, we now show that the NTD of NR2B, in contrast to NR2C and NR2D, also binds zinc, but with a lower affinity. Furthermore, we present evidence that zinc and ifenprodil compete for an overlapping binding site. This modulatory binding site accounts for the submicromolar zinc inhibition of NR1/NR2B receptors. Given that zinc is accumulated and released at many glutamatergic synapses in the CNS, these findings suggest that zinc is the endogenous ligand of the NTD of both NR2A and NR2B, the two major NR2 subunits. Thus, NMDA receptors contain zinc sensors capable of detecting extracellular zinc over a wide concentration range depending on their NR2 subunit composition. The coexistence of subunit-specific zinc-binding sites of high (nanomolar) and low (micromolar) affinity on NMDA receptors raises the possibility that zinc exerts both a tonic and a phasic control of membrane excitability.

Identification of critical residues in the amino terminal domain of the human NR2B subunit involved in the RO 25-6981 binding pocket.

N-Methyl-d-aspartate (NMDA) receptors play key roles in both physiological processes, particularly synaptic plasticity, and in neuropathological states such as epilepsy and acute neurodegeneration. R-(R*,S*)-alpha-(4-Hydroxyphenyl)-beta-methyl-4-(phenyl-methyl)-1-piperidine propanol (RO 25-6981), is a high-affinity and selective blocker of NMDA receptors containing the NR2B subunit. Using site-directed mutagenesis, [3H]RO 25-6981 binding, Xenopus oocyte voltage-clamp recordings, and molecular modeling, we have identified several critical residues involved in the RO 25-6981 binding site within the N-terminal LIVBP-like domain of the human NR2B subunit. Two mutations, NR2B(D101A) and NR2B(F176A), resulted in a complete loss of [3H]RO 25-6981 binding and also abolished the high-affinity RO 25-6981-mediated inhibition of NMDA-induced currents. The mutation NR2B(T233A) led to a marked reduction in binding affinity by 13-fold. Mutations F182A, D104A, or K234A had a more moderate influence on the binding affinity (KD values increased by 8-, 7-, and 6-fold, respectively). In a three-dimensional model of the NR2B LIVBP-like domain based on the X-ray crystal structure of the amino-terminal domain of the mGlu1 receptor, the critical residues are located in the central cleft where interaction with RO 25-6981 may stabilize the closed structure of the domain. Our results suggest that the three amino acids Asp-101, Phe-176, and Thr-233 are important molecular determinants for the high-affinity binding of RO 25-6981 to the LIVBP-like domain of human NR2B. A possible binding mode for RO 25-6981 is proposed.

Mapping the binding site of the neuroprotectant ifenprodil on NMDA receptors.

Ifenprodil is a noncompetitive antagonist of NMDA receptors highly selective for the NMDA receptor 2B (NR2B) subunit. It is widely used as a pharmacological tool to discriminate subpopulations of NMDA receptors, and derivatives are currently being developed as candidate neuroprotectants. Despite numerous studies on the mechanism of action of ifenprodil on NMDA receptors, the structural determinants responsible for the subunit selectivity have not been identified. By combining functional studies on recombinant NMDA receptors and biochemical studies on isolated domains, we now show that ifenprodil binds to the N-terminal leucine/isoleucine/valine-binding protein (LIVBP)-like domain of NR2B. In this domain, several residues, both hydrophilic and hydrophobic, were found to control ifenprodil inhibition. Their location in a modeled three-dimensional structure suggests that ifenprodil binds in the cleft of the LIVBP-like domain of NR2B by a mechanism (Venus-flytrap) resembling that of the binding of Zn on the LIVBP-like domain of NR2A. These results reinforce the proposal that the LIVBP-like domains of NMDA receptors, and possibly of other ionotropic glutamate receptors, bind modulatory ligands. Moreover, they identify the LIVBP-like domain of the NR2B subunit as a promising therapeutic target and provide a framework for designing structurally novel NR2B-selective antagonists.