Structural determinants for activity of the antidepressant vortioxetine at human and rodent 5-HT 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-HT receptor (5-HTR), which features two properties: VTX acts differently on rodent and human 5-HTR, 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-HTR and an agonist-bound-like state of human 5-HTR, in line with the functional profile of the drug.

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 β1-β2 and M2-M3 loops, with agonist binding re-positioning a key β1-β2 glutamate/valine to facilitate the outward motions of a conserved M2-M3 proline to open the channel gate.

Superiority of the Triple-Acting 5-HTR/5-HTR Antagonist and MAO-B Reversible Inhibitor over 5-HTR 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-HTR antagonism and interaction with 5-HTR were determined using molecular dynamic simulations and cryo-electron microscopy, respectively.

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.

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.

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.

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 LGICs for biosensing applications is technically challenging. We have previously designed modified LGICs by linking G protein-coupled receptors (GPCRs) to the Kir6.

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.

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.

Conformational transitions of the serotonin 5-HT receptor.

The serotonin 5-HT 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 membrane; 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-HT receptors are therapeutic targets for emesis and nausea, irritable bowel syndrome and depression.

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.

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-β-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.

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.

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.

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.

Structural approaches of the mitochondrial carrier family.

The transport of solutes across the inner mitochondrial membrane is highly selective and necessitates membrane proteins mainly from the mitochondrial carrier family (MCF). These carriers are required for the transport of a variety of metabolites implicated in all the important processes occurring within the mitochondrial matrix. Due to its high abundance, the ADP/ATP carrier (AAC) is the member of the family that was studied most.

One-microsecond molecular dynamics simulation of channel gating in a nicotinic receptor homologue.

Recently discovered bacterial homologues of eukaryotic pentameric ligand-gated ion channels, such as the Gloeobacter violaceus receptor (GLIC), are increasingly used as structural and functional models of signal transduction in the nervous system. Here we present a one-microsecond-long molecular dynamics simulation of the GLIC channel pH stimulated gating mechanism. The crystal structure of GLIC obtained at acidic pH in an open-channel form is equilibrated in a membrane environment and then instantly set to neutral pH.

Atomic structure and dynamics of pentameric ligand-gated ion channels: new insight from bacterial homologues.

Pentameric ligand-gated ion channels (pLGICs) are widely expressed in the animal kingdom and are key players of neurotransmission by acetylcholine (ACh), gamma-amminobutyric acid (GABA), glycine and serotonin. It is now established that this family has a prokaryotic origin, since more than 20 homologues have been discovered in bacteria. In particular, the GLIC homologue displays a ligand-gated ion channel function and is activated by protons.