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

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 fluorescence intensity and anisotropy upon specific binding to the receptor yielded dissocn. consts. of .apprx.0.2 nM. Fluorescence measurements showed that selective 5HT3 receptor ligands competed for GR-flu binding with a rank order of potency identical to that established with the radioligand [3H]-GR65630. The kinetics of GR-flu binding to the 5HT3 receptor revealed a bimol. assocn. process with an on-rate const. of 1.17106 s-1 M-1 and a biphasic dissocn. reaction with off-rate consts. of 27510-6 and 43*10-6 s-1. The temp. dependence of the dissocn. const. yielded an enthalpic term of -26 kJ mol-1 and an entropic term of 94 J K-1 mol-1 for the binding of GR-flu to the receptor, indicating that both quantities contribute equally to the reaction. An activation enthalpy DH#on and entropy DS#on of binding of 50 kJ mol-1 and 43 J mol-1 K-1 were obtained, indicating that the entropy facilitates the initial steps of GR-flu binding to the 5HT3 receptor. The fluorescence anisotropy of receptor-bound GR-flu and the environmental sensitivity of the fluorescent probe suggest that the binding site has a wide entrance and that it is 0.8 pH unit more acidic than the bulk soln. [on SciFinder (R)]

In vitro and in vivo ligand binding to the 5HT3 serotonin receptor characterised by time-resolved fluorescence spectroscopy

Rudolf Hovius, Horst Pick, Horst Vogel

The binding of the fluorescein-labeled antagonist GR-flu ([1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino- (N-fluoresceinthiocarbamoyl)propyl)-4H-carbazol-4-one]) to a purified, detergent-solubilized ligand-gated ion channel, the type-3 serotonin (5-hydroxytryptamine, 5HT) receptor (5HT3R), was characterized by frequency-domain time-resolved fluorescence spectroscopy (TRFS). Detailed understanding of how ligands interact with the homopentameric receptor was obtained. While a 1:1 stoichiometry was obsd. for the GR-flu - receptor complex, the agonist quipazine bound cooperatively to the receptor, suggesting multiple binding sites for this ligand. The GR-flu-binding site of the receptor was proven to provide an acidic environment as shown by detg. the fraction of bound GR-flu in the protonated state. Fluorescence anisotropy relaxation expts. indicated a hindered but still high mobility for the receptor-bound GR-flu. Hence, the binding site is expected to present a wide opening to the ligand. Finally, the authors succeeded in measuring the binding of GR-flu to 5HT3 receptors in live cells. These results show that the purified and the native receptor behave identically and demonstrate that time-resolved fluorescence measurements are suited to selectively investigate biomol. interactions in live cells. [on SciFinder (R)]

2001

Noninvasive imaging of 5-HT3 receptor trafficking in live cells: From biosynthesis to endocytosis

Cédric Deluz, Erwin Ilegems, Horst Pick, Horst Vogel

Sequential stages in the life cycle of the ionotropic 5-HT3 receptor (5-HT3R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT3R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT3R-specific antagonist. After novel protein synthesis was chem. blocked, the 5-HT3 R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quant. to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT3 receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 mm/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT3 receptors was obsd. within minutes after exposure to an extracellular agonist. The authors' orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking. [on SciFinder (R)]

2004

Structure and function of the nicotinic acetylcholine receptor

Jörg Grandl

The subject of this thesis is the study of the structure and function of the human muscle nicotinic acetylcholine receptor (nAChR), which is a typical member of the large class of ligand-gated ion channels. Appropriate techniques, like site-directed mutagenesis, patch-clamp and fluorescence microscopy, and combinations thereof, were applied to gain insight into selected aspects of the structure-function relationship of this receptor. Special emphasis was put on the investigation of single receptors, wherever this promised scientific benefit. Experiments with the aim to study structural rearrangements of nAChR upon ligand binding by fluorescence resonance energy transfer (FRET) were undertaken. Such intramolecular motions are expected to link ligand binding to channel gating and are therefore fundamental to understand the function of nAChR. Double-cysteine mutants were produced and receptors were labelled on living cells in order to measure changes in FRET efficiency due to ligand binding. A novel class of functionally modified ligands for the nAChR was characterized by patch-clamp technique. Derivatives that carry a thiol-reactive group were shown to be full and very potent agonists. On the bases of a structural model of the binding site, residues were selected, that are likely to be in contact with the bound ligand. Equivalent single-cysteine mutants were produced to covalently bind these agonists and thereby to gain information about the mechanisms of ligand binding and receptor activation. Ligands that were coupled to fluorophores turned out to be either partial or full agonists with nanomolar efficacies for the nAChR. Their use to study the diffusion of single nAChR on living HEK293-cells for different functional states is described in detail. The dependency of the lateral diffusion on the activation state (closed/opened/desensitized) was studied. Additionally these ligands served to demonstrate the feasibility to simultaneously combine patch-clamp and fluorescent binding experiments on a single-molecule level. Acetylcholine receptors carrying a single point-mutation, related to the congenital myasthenic syndrome, were found to stabilize an intermediate conductance state and were studied in detail by single-channel electrophysiology. The dependency of the gating behavior on different ligands, ligand concentration and transmembrane voltage was investigated. Mutants, carrying equivalent mutations on different subunits were produced to reveal the origin of this effect. Quantitative characterization was obtained by dwell-time and spectral noise-analysis, in order to gain insight on sub-millisecond fluctuations of the channel lining transmembrane regions of the nAChR and the gating mechanism in general.

EPFL2005

Structure and function of the nicotinic acetylcholine receptor

Jörg Grandl

EPFL2005