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

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-nitrobenz-2-oxa-1,3-diazol-4-yl], and the cyanine dye Cy5. These labels cover a large part of the visible electromagnetic spectrum. It is shown that the photophysical and chemical properties have a direct influence on the measurement quality (duration of measurement, signal-to-noise ratio) and the ligand-receptor interactions (dissociation constants), respectively. This makes it necessary to choose a suitable label or a combination of labels for receptor studies. The affinities of the fluorescently labeled ligands determined by FCS were virtually identical to the values obtained by radioligand binding experiments. Moreover, the dissociation constant of a nonfluorescent receptor ligand was determined successfully by an FCS competition assay. The experimental results showed that only one antagonist binds to the receptor, in agreement with measurements previously published [Tairi et al. (1998) Biochemistry 37, 15850-15864].

Biological samples studied by optical nanospectroscopy

Johanna Generosi

The different parts of the electromagnetic spectrum result in diverse effects upon interaction with matter: according to the wavelength, the radiation has energy appropriate for the excitation of a specific physical process. X-rays can be used as a tool to analyze the structure of matter since their wavelength is comparable with the interatomic distances. Infrared light is in the spectral region that excites molecular vibrations and is employed to investigate the chemical composition of a material. Visible radiation can study the optical properties of a sample, such as the fluorescence and the absorbance, and provide a chemical fingerprint when the inelastically scattered light is detected. In this thesis work these light sources are used in diverse experimental approaches to study structured biological specimens, resulting in a detailed chemical and physical characterization at the atomic and molecular scale. Conventional spectroscopy is often not enough sensitive and spatially resolved to detect specific elements or domains in a sample. The need of imaging objects on increasingly finer scales and spatially localize specific molecules, brought to combine infrared, visible and Raman spectroscopy with scanning near-field microscopy giving rise to a powerful nanospectroscopic tool used to perform simultaneous topographical measurements and optical/chemical characterizations with subwavelength resolution, overcoming the diffraction limit of light. Our study combines X-ray diffraction and reflectivity with optical nanospectroscopy to investigate the order and clustering of lipid bilayers, the interaction between solid-supported membranes and embedded alamethicin peptides, the optical and chemical properties of hippocampal neuron cells and the trafficking mechanism of specific neuron receptors.

EPFL2009

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

Marinela Gjoni Boillat, Rudolf Hovius, Christoph Schreiter, Jean-Manuel Segura, Horst Vogel

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 which the ligand associates with the receptor and then dissociates; and then repeatedly monitoring the fluorescence of the cell. USE - In a screening procedure for the identification and/or characterization of pharmaceutical drug candidate molecules and for determining a receptor-ligand interaction to allow sensitive measurements on single cells carried out in a microfluidic structure or on a chip (claimed) and can be used to characterize single or multiple cells by determining the presence of receptors or other proteins on the surface of the cells e.g. native cells, multiple cells like tissue sections, genetically modified cells or with cells immobilized on a solid surface, or in a matrix like a gel, or with cells in a liquid medium. ADVANTAGE - The method enables pharmacological investigations to be performed on single cells expressing the natural amount of receptor, which usually is very difficult due to photobleaching. The invention performs fluorescence binding assays, the reversible sequential binding assay that solves problems associated with previous measurements of ligand-receptor interactions by applying repetitively-completely reversible and specific fluorescently labeled ligands with fast association-dissociation kinetics. The new approach overcomes problems related to photobleaching so that cells expressing low amounts of receptors down to the single-molecule level can be investigated. The high sensitivity of the method allows the detection of little abundant proteins, thus enabling characterization of native cells. It can be applied even in the case of strong photobleaching opening the possibility to investigate single cells with low receptor concentrations comparable to native conditions in tissue cells. Furthermore, the influence of stimuli, such as ligands, can be rapidly and repetitively investigated using our reversible binding assay. A key advantage of the measurement approach is that pharmacological investigations can be performed on single cells expressing the natural amount of receptor, which usually is very difficult due to photobleaching. Using repetitive measurements, binding parameters can be extracted and investigations can be performed even when photobleaching becomes extremely strong as it is the case at lowest expression levels. Ultimately, single-molecule sensitivity can be achieved as illustrated on the nAChR. This opens the possibility to perform pharmacological investigations on tissue cells expressing minimal amounts of membrane receptors. By performing repetitive measurements, tenth of image sequences can be acquired to collect extensive statistics within a single experiment. High-throughput screening of potential drugs can be performed on a single cell with a rate only depending on the association-dissociation cycle time, consuming only minimal amount of testing components. One can expect that the use of ligands with faster kinetics could reduce this time down to 5 minutes. In addition, the use of more complex microstructures enables complete binding assays on a single cell or increasing statistics by parallel approaches using only some hundred nano liters of cell suspension. This way, screening more than 250 ligands per day using one single cell extracted from an animal tissue or obtained from a body fluid is a realistic goal.

2006

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.