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.

A release of local subunit conformational heterogeneity underlies gating in a muscle nicotinic acetylcholine receptor.

Synaptic receptors respond to neurotransmitters by opening an ion channel across the post-synaptic membrane to elicit a cellular response. Here we use recent Torpedo acetylcholine receptor structures and functional measurements to delineate a key feature underlying allosteric communication between the agonist-binding extracellular and channel-gating transmembrane domains. Extensive mutagenesis at this inter-domain interface re-affirms a critical energetically coupled role for the principal α subunit ß1-ß2 and M2-M3 loops, with agonist binding re-positioning a key ß1-ß2 glutamate/valine to facilitate the outward motions of a conserved M2-M3 proline to open the channel gate. Notably, the analogous structures in non-α subunits adopt a locally active-like conformation in the apo state even though each L9' hydrophobic gate residue in each pore-lining M2 α-helix is closed. Agonist binding releases local conformational heterogeneity transitioning all five subunits into a conformationally symmetric open state. A release of conformational heterogeneity provides a framework for understanding allosteric communication in pentameric ligand-gated ion channels.

Superiority of the Triple-Acting 5-HT6R/5-HT3R Antagonist and MAO-B Reversible Inhibitor PZ-1922 over 5-HT6R Antagonist Intepirdine in Alleviation of Cognitive Deficits in Rats.

The multifactorial origin and neurochemistry of Alzheimer's disease (AD) call for the development of multitarget treatment strategies. We report a first-in-class triple acting compound that targets serotonin type 6 and 3 receptors (5-HT-Rs) and monoamine oxidase type B (MAO-B) as an approach for treating AD. The key structural features required for MAO-B inhibition and 5-HT6R antagonism and interaction with 5-HT3R were determined using molecular dynamic simulations and cryo-electron microscopy, respectively. Bioavailable PZ-1922 reversed scopolamine-induced cognitive deficits in the novel object recognition test. Furthermore, it displayed superior pro-cognitive properties compared to intepirdine (a 5-HT6R antagonist) in the AD model, which involved intracerebroventricular injection of an oligomeric solution of amyloid-ß peptide (oAß) in the T-maze test in rats. PZ-1922, but not intepirdine, restored levels of biomarkers characteristic of the debilitating effects of oAß. These data support the potential of a multitarget approach involving the joint modulation of 5-HT6R/5-HT3R/MAO-B in AD.

Role of surfactants in electron cryo-microscopy film preparation.

Single-particle electron cryo-microscopy (cryo-EM) has become an effective and straightforward approach to determine the structure of membrane proteins. However, obtaining cryo-EM grids of sufficient quality for high-resolution structural analysis remains a major bottleneck. One of the difficulties arises from the presence of detergents, which often leads to a lack of control of the ice thickness. Amphipathic polymers such as amphipols (APols) are detergent substitutes, which have proven to be valuable tools for cryo-EM studies. In this work, we investigate the physico-chemical behavior of APol- and detergent-containing solutions and show a correlation with the properties of vitreous thin films in cryo-EM grids. This study provides new insight on the potential of APols, allowing a better control of ice thickness while limiting protein adsorption at the air-water interface, as shown with the full-length mouse serotonin 5-HT3A receptor whose structure has been solved in APol. These findings may speed up the process of grid optimization to obtain high-resolution structures of membrane proteins.

The molecular mechanism of snake short-chain α-neurotoxin binding to muscle-type nicotinic acetylcholine receptors.

Bites by elapid snakes (e.g. cobras) can result in life-threatening paralysis caused by venom neurotoxins blocking neuromuscular nicotinic acetylcholine receptors. Here, we determine the cryo-EM structure of the muscle-type Torpedo receptor in complex with ScNtx, a recombinant short-chain α-neurotoxin. ScNtx is pinched between loop C on the principal subunit and a unique hairpin in loop F on the complementary subunit, thereby blocking access to the neurotransmitter binding site. ScNtx adopts a binding mode that is tilted toward the complementary subunit, forming a wider network of interactions than those seen in the long-chain α-Bungarotoxin complex. Certain mutations in ScNtx at the toxin-receptor interface eliminate inhibition of neuronal α7 nAChRs, but not of human muscle-type receptors. These observations explain why ScNtx binds more tightly to muscle-type receptors than neuronal receptors. Together, these data offer a framework for understanding subtype-specific actions of short-chain α-neurotoxins and inspire strategies for design of new snake antivenoms.

Conformational transitions and ligand-binding to a muscle-type nicotinic acetylcholine receptor.

Fast synaptic communication requires receptors that respond to the presence of neurotransmitter by opening an ion channel across the post-synaptic membrane. The muscle-type nicotinic acetylcholine receptor from the electric fish, Torpedo, is the prototypic ligand-gated ion channel, yet the structural changes underlying channel activation remain undefined. Here we use cryo-EM to solve apo and agonist-bound structures of the Torpedo nicotinic receptor embedded in a lipid nanodisc. Using both a direct biochemical assay to define the conformational landscape and molecular dynamics simulations to assay flux through the pore, we correlate structures with functional states and elucidate the motions that lead to pore activation of a heteromeric nicotinic receptor. We highlight an underappreciated role for the complementary subunit in channel gating, establish the structural basis for the differential agonist affinities of α/δ versus α /γ sites, and explain why nicotine is less potent at muscle nicotinic receptors compared to neuronal ones.

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM.

Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.

Distinct classes of potassium channels fused to GPCRs as electrical signaling biosensors.

Ligand-gated ion channels (LGICs) are natural biosensors generating electrical signals in response to the binding of specific ligands. Creating de novo LGICs for biosensing applications is technically challenging. We have previously designed modified LGICs by linking G protein-coupled receptors (GPCRs) to the Kir6.2 channel. In this article, we extrapolate these design concepts to other channels with different structures and oligomeric states, namely a tetrameric viral Kcv channel and the dimeric mouse TREK-1 channel. After precise engineering of the linker regions, the two ion channels were successfully regulated by a GPCR fused to their N-terminal domain. Two-electrode voltage-clamp recordings showed that Kcv and mTREK-1 fusions were inhibited and activated by GPCR agonists, respectively, and antagonists abolished both effects. Thus, dissimilar ion channels can be allosterically regulated through their N-terminal domains, suggesting that this is a generalizable approach for ion channel engineering.

International Union of Basic and Clinical Pharmacology. CX. Classification of Receptors for 5-hydroxytryptamine; Pharmacology and Function.

5-HT receptors expressed throughout the human body are targets for established therapeutics and various drugs in development. Their diversity of structure and function reflects the important role 5-HT receptors play in physiologic and pathophysiological processes. The present review offers a framework for the official receptor nomenclature and a detailed understanding of each of the 14 5-HT receptor subtypes, their roles in the systems of the body, and, where appropriate, the (potential) utility of therapeutics targeting these receptors. SIGNIFICANCE STATEMENT: This review provides a comprehensive account of the classification and function of 5-hydroxytryptamine receptors, including how they are targeted for therapeutic benefit.

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.

A lipid site shapes the agonist response of a pentameric ligand-gated ion channel.

Phospholipids are key components of cellular membranes and are emerging as important functional regulators of different membrane proteins, including pentameric ligand-gated ion channels (pLGICs). Here, we take advantage of the prokaryote channel ELIC (Erwinia ligand-gated ion channel) as a model to understand the determinants of phospholipid interactions in this family of receptors. A high-resolution structure of ELIC in a lipid-bound state reveals a phospholipid site at the lower half of pore-forming transmembrane helices M1 and M4 and at a nearby site for neurosteroids, cholesterol or general anesthetics. This site is shaped by an M4-helix kink and a Trp-Arg-Pro triad that is highly conserved in eukaryote GABAA/C and glycine receptors. A combined approach reveals that M4 is intrinsically flexible and that M4 deletions or disruptions of the lipid-binding site accelerate desensitization in ELIC, suggesting that lipid interactions shape the agonist response. Our data offer a structural context for understanding lipid modulation in pLGICs.

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.

Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor.

There is growing interest in the use of mammalian protein expression systems, and in the use of antibody-derived chaperones, for structural studies. Here, we describe protocols ranging from the production of recombinant membrane proteins in stable inducible cell lines to biophysical characterization of purified membrane proteins in complex with llama antibody domains. These protocols were used to solve the structure of the mouse 5-HT3 serotonin receptor but are of broad applicability for crystallization or cryo-electron microscopy projects.

X-ray structure of the mouse serotonin 5-HT3 receptor.

Neurotransmitter-gated ion channels of the Cys-loop receptor family mediate fast neurotransmission throughout the nervous system. The molecular processes of neurotransmitter binding, subsequent opening of the ion channel and ion permeation remain poorly understood. Here we present the X-ray structure of a mammalian Cys-loop receptor, the mouse serotonin 5-HT3 receptor, at 3.5 Å resolution. The structure of the proteolysed receptor, made up of two fragments and comprising part of the intracellular domain, was determined in complex with stabilizing nanobodies. The extracellular domain reveals the detailed anatomy of the neurotransmitter binding site capped by a nanobody. The membrane domain delimits an aqueous pore with a 4.6 Å constriction. In the intracellular domain, a bundle of five intracellular helices creates a closed vestibule where lateral portals are obstructed by loops. This 5-HT3 receptor structure, revealing part of the intracellular domain, expands the structural basis for understanding the operating mechanism of mammalian Cys-loop receptors.

Sedimentation velocity analytical ultracentrifugation in hydrogenated and deuterated solvents for the characterization of membrane proteins.

This chapter is a step-by-step protocol for setting up, realizing, and analyzing sedimentation velocity experiments in hydrogenated and deuterated solvents, in the context of the characterization of membrane protein, in terms of homogeneity, association state, and amount of bound detergent, based on a real case study of the membrane protein BmrA solubilized in n-Dodecyl-ß-D-Maltopyranoside) detergent.

Large scale expression and purification of the mouse 5-HT3 receptor.

Receptors of the Cys-loop family are central to neurotransmission and primary therapeutic targets. In order to decipher their gating and modulation mechanisms, structural data is essential. However, structural studies require large amounts of pure, functional receptors. Here, we present the expression and purification of the mouse serotonin 5-HT3 receptor to high purity and homogeneity levels. Inducible expression in human embryonic kidney 293 cells in suspension cultures with orbital shaking resulted in yields of 6-8mg receptor per liter of culture. Affinity purification using a strep tag provided pure protein in active form. Further deglycosylation and removal of the purification tag led to a pentameric receptor after size-exclusion chromatography, at the milligram scale. This material is suitable for crystallography, as demonstrated by X-ray diffraction of receptor crystals at low resolution.

Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines.

GABA(A) receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABA(A) receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their concentration, occupy two possible sites in ELIC. An intrasubunit site is adjacent to the GABA-recognition site but faces the channel vestibule. A second intersubunit site partially overlaps with the GABA site and likely corresponds to a low-affinity benzodiazepine-binding site in GABA(A) receptors that mediates inhibitory effects of the benzodiazepine flurazepam. Our study offers a structural view how GABA and benzodiazepines are recognized at a GABA-activated ion channel.

A locally closed conformation of a bacterial pentameric proton-gated ion channel.

Pentameric ligand-gated ion channels mediate signal transduction through conformational transitions between closed-pore and open-pore states. To stabilize a closed conformation of GLIC, a bacterial proton-gated homolog from Gloeobacter violaceus whose open structure is known, we separately generated either four cross-links or two single mutations. We found all six mutants to be in the same 'locally closed' conformation using X-ray crystallography, sharing most of the features of the open form but showing a locally closed pore as a result of a concerted bending of all of its M2 helices. The mutants adopt several variant conformations of the M2-M3 loop, and in all cases an interacting lipid that is observed in the open form disappears. A single cross-linked mutant is functional, according to electrophysiology, and the locally closed structure of this mutant indicates that it has an increased flexibility. Further cross-linking, accessibility and molecular dynamics data suggest that the locally closed form is a functionally relevant conformation that occurs during allosteric gating transitions.

X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel.

General anaesthetics have enjoyed long and widespread use but their molecular mechanism of action remains poorly understood. There is good evidence that their principal targets are pentameric ligand-gated ion channels (pLGICs) such as inhibitory GABA(A) (γ-aminobutyric acid) receptors and excitatory nicotinic acetylcholine receptors, which are respectively potentiated and inhibited by general anaesthetics. The bacterial homologue from Gloeobacter violaceus (GLIC), whose X-ray structure was recently solved, is also sensitive to clinical concentrations of general anaesthetics. Here we describe the crystal structures of the complexes propofol/GLIC and desflurane/GLIC. These reveal a common general-anaesthetic binding site, which pre-exists in the apo-structure in the upper part of the transmembrane domain of each protomer. Both molecules establish van der Waals interactions with the protein; propofol binds at the entrance of the cavity whereas the smaller, more flexible, desflurane binds deeper inside. Mutations of some amino acids lining the binding site profoundly alter the ionic response of GLIC to protons, and affect its general-anaesthetic pharmacology. Molecular dynamics simulations, performed on the wild type (WT) and two GLIC mutants, highlight differences in mobility of propofol in its binding site and help to explain these effects. These data provide a novel structural framework for the design of general anaesthetics and of allosteric modulators of brain pLGICs.