Publications by Rudolf Hovius | EPFL Graph Search

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Since the first description of 5-HT3 receptors more than 50 years ago, there has been speculation about the molecular basis of their receptor heterogeneity. We have cloned the genes encoding novel 5-HT3 subunits 5-HT3C, 5-HT3D, and 5-HT3E and have shown th ...

NOVELTY - Determining a receptor-ligand interaction involves repeatedly contacting a cell comprising a receptor, with a ligand, of the receptor in which the ligand has a fluorescence labeling group and binds reversibly to the receptor under conditions in w ...

The application of fluorescent receptor ligands has become widespread, incited by two important reasons. “Seeing is believing”—it is possible to visualize in real time in live cells ligand–receptor interactions, and to locate the receptors with subcellular ...

The lateral organization of a prototypical G protein-coupled receptor, the neurokinin-1 receptor (NK1R), was investigated in living cells by fluorescence resonance energy transfer (FRET) microscopy, taking advantage of the recently developed acyl carrier p ...

Nicotinic acetylcholine receptors (nAChR) in muscle fibers are densely packed in the postsynaptic region at the neuromuscular junction. Rapsyn plays a central role in directing and clustering nAChR during cellular differentiation and neuromuscular junction ...

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that switches upon activation from a closed state to a full conducting state. We found that the mutation delta S268K, located at 12' position of the second transmembrane domain of t ...

Cellular signalling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show how cell-derived vesicles can be used for anlaysing transmembrane signalling. The vesicles are derived from live mammalian cells by using either chemicals, or by optical tweezers and they comprise parts of the plasma membrane and cytosol of the mother cell. We measured in vesicles derived from individual cells with single molecule sensitivity the different step

The thermodn. and kinetics of ligand binding to the purified serotonin 5HT3 receptor and the local environment of the bound ligand were studied by fluorescence spectroscopy using a novel fluorescein-labeled ligand GR-flu [1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino-(N-fluorescein-thiocarbamoyl)-propyl)-4H-carbazol-4-one]. Electrophysiol. investigations demonstrated GR-flu to be an antagonist, and radioligand competition assays delivered a dissocn. const. of 0.32 nM. Changes in the fl

The disulfide bonds that form between two cysteine residues are important in defining and rigidifying the structures of proteins and peptides. In polypeptides containing multiple cysteine residues, disulfide isomerization can lead to multiple products with ...

The expression of recombinant proteins with the Semliki Forest Virus (SFV) system has been scaled up to bioreactor scale. As a model protein for this study the human 5-HT(3) receptor was chosen. The gene for the receptor was subcloned into the SFV expression plasmid pSFV1. Virus production by in vivo packaging and production of the recombinant protein was scaled up, the latter to a reactor volume of 11.5 l. A Vibromix(TM) agitation system was chosen to overcome aggregation problems of BHK cells in suspensio

The ionotropic 5HT3 receptor was expressed in transiently transfected mammalian cells, yielding an unprecedented high concn. of up to 12 million receptors per cell. Receptor traffic in the plasma membrane of live cells was obsd. continuously over 24 h by fluorescence scanning confocal microscopy. This was possible by using 5HT3 receptor-specific fluorescent ligands with high binding affinity and low off-rate to pulse label receptors at any time after appearance on the cell surface, and label subsequently th

The nicotinic acetylcholine receptor from Torpedo was immobilised in tethered membranes. Surface plasmon resonance was used to quantify the binding of ligands and antibodies to the receptor. The orientation and structural integrity of the surface-reconstituted receptor was probed using monoclonal antibodies, demonstrating that approximately 65% of the receptors present their ligand-binding site towards the lumen of the flow cell and that at least 85% of these receptors are structurally intact. The conformat

G protein-coupled receptors (GPCRs) constitute a large class of seven transmembrane proteins, which bind selectively agonists or antagonists with important consequences for cellular signaling and function. Comprehension of the mol. details of ligand binding is important for the understanding of receptor function and in turn for the design and development of novel therapeutic compds. Here we show how ligand-receptor interaction can be investigated in situ with high sensitivity on sensor surfaces by total int

The DNA sensor cGAS initiates innate immune responses following microbial infection, cellular stress, and cancer1. Upon activation by double-stranded DNA, cytosolic cGAS produces 2’3’ cyclic GMP-AMP and triggers inflammatory cytokine and type I interferon ...

The 5-hydroxytryptamine receptor of type 3 was investigated by fluorescence correlation spectroscopy (FCS). Binding constants of fluorescently labeled ligands, the stoichiometry, and the mass of the receptor are readily accessible by this technique, while the duration of measurement is on the order of seconds to minutes. The receptor antagonist 1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-aminopropyl)-4H-carbazol-4-one (GR-H) was labeled with the fluorophores rhodamine 6G, fluorescein, N-[7

Ligand-gated ion channels of the Cys loop family are receptors for small amine-contg. neurotransmitters. Charged amino acids are strongly conserved in the ligand-binding domain of these receptor proteins. To investigate the role of particular residues in ligand binding of the serotonin 5-HT3AS receptor (5-HT3R), glutamate amino acid residues at three different positions, Glu97, Glu224, and Glu235, in the extracellular N-terminal domain were substituted with aspartate and glutamine using site-directed mutage

Sensitive live-cell fluorescence microscopy and single-molecule imaging are severely limited by rapid photobleaching of fluorescent probes. Herein, we show how to circumvent this problem using a novel, generic labeling strategy. Small nickel-nitrilotriacet ...

NOVELTY - Conjugate (I) of an agonist ligand directed to ligand-gated ion channels (LGICs) of the Cys loop-family with a chromophore, is new. USE - (I) is useful in the receptor proteins and/or their interaction with the agonistic ligand, where the interac ...

Nicotinic acetylcholine receptors are heteropentameric ion channels that open upon activation to a single conducting state. The second transmembrane segments of each subunit were identified as channel-forming elements, but their respective contribution in ...

The highly efficient Semliki Forest virus (SFV) system has been applied to express several recombinant 7TM receptors and ligand-gated ion channels. High affinity binding and functional coupling to G proteins were obtained for the receptors. The ligand-gated ion channels showed high specific binding activity and high-amplitude electrophysiol. responses in whole-cell patch clamp recordings. The rapid and simple generation of high titer virus stocks has made the SFV system also useful for site-directed mutagen

Rudolf Hovius

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Serotonin receptor diversity in the human colon: Expression of serotonin type 3 receptor subunits 5-HT3C, 5-HT3D, and 5-HT3E

Determining a receptor-ligand interaction useful in e.g. drug screening involves repetitive application of reversibly binding fluorescent ligands with fast association-dissociation kinetics

Characterization and Validation of Fluorescent Receptor Ligands: A Case Study of the Ionotropic Serotonin Receptor

FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells

Acetylcholine Receptor Organization in Membrane Domains in Muscle Cells: EVIDENCE FOR RAPSYN-INDEPENDENT AND RAPSYN-DEPENDENT MECHANISMS

Modulation of proton-induced current fluctuations in the human nicotinic acetylcholine receptor channel

Cell-derived plasma membrane vesicles as minimal cells for analyzing transmembrane signaling

Ligand binding to the serotonin 5HT3 receptor studied with a novel fluorescent ligand

Dithiol amino acids can structurally shape and enhance the ligand-binding properties of polypeptides

Large scale transient 5-HT3 receptor production with the Semliki Forest Virus Expression System

Monitoring expression and clustering of the ionotropic 5HT3 receptor in plasma membranes of live biological cells

Functional immobilisation of the nicotinic acetylcholine receptor in tethered lipid membranes

Ligand Binding to G Protein-Coupled Receptors in Tethered Cell Membranes

Structural mechanism of cGAS inhibition by the nucleosome

Study of ligand-receptor interactions by fluorescence correlation spectroscopy with different fluorophores: Evidence that the homopentameric 5-hydroxytryptamine type 3(As) receptor binds only one ligand

Characterization of the Ligand-binding Site of the Serotonin 5-HT3 Receptor: The Role of Glutamate Residues 97, 224,and 235

Repetitive Reversible Labeling of Proteins at Polyhistidine Sequences for Single-Molecule Imaging in Live Cells

New conjugate of agonist ligand directed to ligand-gated ion channels of cysteine loop-family with chromophore, useful, e.g. in receptor proteins/their interaction with agonistic ligand and to study the effect of unlabelled ligands

Functional asymmetry of transmembrane segments in nicotinic acetylcholine receptors

The Semliki Forest virus system: receptor expression and scale-up