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.

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.

Two-dimensional crystallization of the mouse serotonin 5-HT3A receptor.

The mouse serotonin 5-HT3A receptor is a homo-pentameric ligand-gated ion channel (pLGIC) mediating fast excitatory neurotransmission in the central nervous system. The molecular mechanism of ion permeation of 5-HT3A receptors triggered by the neurotransmitter serotonin is not yet fully understood. The recent X-ray structure of the mouse serotonin 5-HT3A receptor in complex with a stabilizing nanobody revealed for the first time the entire structure of a mammalian pLGIC in detergent. Structural information of the receptor in a lipid bilayer however is still limited primarily due to the lack of 2D crystals of the receptor in a lipid bilayer. Here we present our results on the formation and improvement of diffracting 2D crystals of the mouse 5-HT3A by limited proteolysis and addition of conformational nanobodies.

The Structure of the Mouse Serotonin 5-HT3 Receptor in Lipid Vesicles.

The function of membrane proteins is best understood if their structure in the lipid membrane is known. Here, we determined the structure of the mouse serotonin 5-HT3 receptor inserted in lipid bilayers to a resolution of 12 Å without stabilizing antibodies by cryo electron tomography and subtomogram averaging. The reconstruction reveals protein secondary structure elements in the transmembrane region, the extracellular pore, and the transmembrane channel pathway, showing an overall similarity to the available X-ray model of the truncated 5-HT3 receptor determined in the presence of a stabilizing nanobody. Structural analysis of the 5-HT3 receptor embedded in a lipid bilayer allowed the position of the membrane to be determined. Interactions between the densely packed receptors in lipids were visualized, revealing that the interactions were maintained by the short horizontal helices. In combination with methodological improvements, our approach enables the structural analysis of membrane proteins in response to voltage and ligand gating.

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.

Downscaling the analysis of complex transmembrane signaling cascades to closed attoliter volumes.

Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity. The vesicles are derived from live mammalian cells using chemicals or optical tweezers. They comprise parts of a cell's plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells. Using fluorescence microscopies, we measured in individual vesicles the different steps of G-protein-coupled receptor mediated signaling like ligand binding to receptors, subsequent G-protein activation and finally arrestin translocation indicating receptor deactivation. Observing cellular signaling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis, and investigating receptor mediated signaling in multiarray format.

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.

Thermal unfolding of a mammalian pentameric ligand-gated ion channel proceeds at consecutive, distinct steps.

Pentameric ligand-gated ion channels (LGICs) play an important role in fast synaptic signal transduction. Binding of agonists to the ß-sheet-structured extracellular domain opens an ion channel in the transmembrane α-helical region of the LGIC. How the structurally distinct and distant domains are functionally coupled for such central transmembrane signaling processes remains an open question. To obtain detailed information about the stability of and the coupling between these different functional domains, we analyzed the thermal unfolding of a homopentameric LGIC, the 5-hydroxytryptamine receptor (ligand binding, secondary structure, accessibility of Trp and Cys residues, and aggregation), in plasma membranes as well as during detergent extraction, purification, and reconstitution into artificial lipid bilayers. We found a large loss in thermostability correlating with the loss of the lipid bilayer during membrane solubilization and purification. Thermal unfolding of the 5-hydroxytryptamine receptor occurred in consecutive steps at distinct protein locations. A loss of ligand binding was detected first, followed by formation of different transient low oligomeric states of receptor pentamers, followed by partial unfolding of helical parts of the protein, which finally lead to the formation receptor aggregates. Structural destabilization of the receptor in detergents could be partially reversed by reconstituting the receptor into lipid bilayers. Our results are important because they quantify the stability of LGICs during detergent extraction and purification and can be used to create stabilized receptor proteins for structural and functional studies.

A zeptoliter volume meter for analysis of single protein molecules.

A central goal in bioanalytics is to determine the concentration of and interactions between biomolecules. Nanotechnology allows performing such analyses in a highly parallel, low-cost, and miniaturized fashion. Here we report on label-free volume, concentration, and mobility analysis of single protein molecules and nanoparticles during their diffusion through a subattoliter detection volume, confined by a 100 nm aperture in a thin gold film. A high concentration of small fluorescent molecules renders the aqueous solution in the aperture brightly fluorescent. Nonfluorescent analytes diffusing into the aperture displace the fluorescent molecules in the solution, leading to a decrease of the detected fluorescence signal, while analytes diffusing out of the aperture return the fluorescence level. The resulting fluorescence fluctuations provide direct information on the volume, concentration, and mobility of the nonfluorescent analytes through fluctuation analysis in both time and amplitude.

Activation of G-protein-coupled receptors in cell-derived plasma membranes supported on porous beads.

G-protein-coupled receptors (GPCRs) are ubiquitous mediators of signal transduction across cell membranes and constitute a very important class of therapeutic targets. In order to study the complex biochemical signaling network coupling to the intracellular side of GPCRs, it is necessary to engineer and control the downstream signaling components, which is difficult to realize in living cells. We have developed a bioanalytical platform enabling the study of GPCRs in their native membrane transferred inside-out from live cells to lectin-coated beads, with both membrane sides of the receptor being accessible for molecular interactions. Using heterologously expressed adenosine A(2A) receptor carrying a yellow fluorescent protein, we showed that the tethered membranes comprised fully functional receptors in terms of ligand and G protein binding. The interactions between the different signaling partners during the formation and subsequent dissociation of the ternary signaling complex on single beads could be observed in real time using multicolor fluorescence microscopy. This approach of tethering inside-out native membranes accessible from both sides is straightforward and readily applied to other transmembrane proteins. It represents a generic platform suitable for ensemble as well as single-molecule measurements to investigate signaling processes at plasma membranes.

Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems.

Production of recombinant receptors has been one of the major bottlenecks in structural biology on G protein-coupled receptors (GPCRs). The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus (SFV) vectors. Evaluation by immunodetection demonstrated that 50% of a total of 103 GPCRs were expressed in bacterial inclusion bodies, 94% in yeast cell membranes and 95% in SFV-infected mammalian cells. The expression levels varied from low to high and the various GPCR families and subtypes were analyzed for their expressability in each expression system. More than 60% of the GPCRs were expressed at milligram levels or higher in one or several systems, compatible to structural biology applications. Functional activity was determined by binding assays in yeast and mammalian cells and the correlation between immunodetection and binding activity was analyzed.

Semliki Forest virus vectors for overexpression of 101 G protein-coupled receptors in mammalian host cells.

Semliki Forest virus vectors were applied for the evaluation of 101 G protein-coupled receptors in three mammalian cell lines. Western blotting demonstrated that 95 of the 101 tested GPCRs showed positive signals. A large number of the GPCRs were expressed at high levels suggesting receptor yields in the range of 1 mg/L or higher, suitable for structural biology applications. Specific binding assays on a selected number of GPCRs were carried out to compare the correlation between total and functional protein expression. Ligands and additives supplemented to the cell culture medium were evaluated for expression enhancement. Selected GPCRs were also expressed from mutant SFV vectors providing enhanced protein expression and reduced host cell toxicity in attempts to further improve receptor yields.