Récepteur nicotinique | EPFL Graph Search

Le récepteur nicotinique de l’acétylcholine est un récepteur ionotrope perméable aux ions sodium ainsi qu'aux ions potassium (canal cationique non spécifique), sensible à l'acétylcholine. Il tient son nom de l'un de ses agonistes, la nicotine, par opposition aux récepteurs muscariniques (récepteurs métabotropes). Il fait partie de la super-famille des récepteurs-canaux nicotinoïdes. Localisation Les récepteurs nicotiniques de l'acétylcholine (nAChRs), en plus de leur rôle primordial dans la transmission neuromusculaire et motrice autonome, sont impliqués dans diverses fonctions au niveau du système nerveux central, en particulier dans le contrôle des mouvements volontaires, la mémoire et l'attention, le sommeil et la veille, la douleur et l'anxiété. Il est principalement présent dans le système nerveux autonome. En effet, dans ce système, composé des systèmes parasympathique et orthosympathique régulant les fonctions basales de l'organisme (la vie végétative), l'acétylcholin

Supported lipid monolayers for the investigation of specific interactions between proteins and ligands

In this work, the specific and nonspecific binding of proteins to planar surfaces was investigated. The surfaces were first modified by the spontaneous adsorption of a number of synthesized 1,2-diacyl-sn-glycero-3-phosphatidylcholine lipids with different ω-mercapto fatty acids. The influence of packing density, order, fluidity, and mobility of the head groups of these supported thiolipid monolayers on the nonspecific adsorption of proteins was systematically investigated. Nonspecific binding of fibrinogen, IgG, BSA, and rabbit serum to such surfaces was monitored on-line by surface plasmon resonance spectroscopy (SPR) and was found to be exceptionally low. Characterization of the supported thiolipid monolayers by grazing incidence infrared spectroscopy, wetting, impedance spectroscopy, calorimetry, and SPR indicates that protein adsorption is influenced by the nature of the fatty acids and suggests that besides the molecular structure of the layer-forming molecules, other supramolecular aspects such as flexibility of the immobilized lipid layer or protrusion of the lipid head groups play a role. These investigations point the way for the construction of even more highly protein repellent surfaces with potential technical applications in the medical (implants) and diagnostic fields (biosensors). To investigate specific protein-surface interactions, acetylcholine containing ligand molecules of the general molecular structure HS(CH2)15(CO)O(CH2CH2O)nCH2(CO)OCH2CH2N+(CH3)3 X- (n = 4, 6, 13) were synthesized. They allow acetylcholine, the natural agonist of the acetylcholine receptor, to be covalently immobilized on a surface. Mixed layers of thiolipids and ligand molecules were formed by self-assembly on planar gold substrates and specific interactions between the immobilized acetylcholine and the nicotinic acetylcholine receptor (nAchR) from Torpedo Californica have were investigated on-line by SPR. Though it seems that the receptor binds specifically, bound receptor could be only partially displaced from the surface by excess free ligand. However, further improvement of the sensor surfaces will surely provide in the future a powerful tool for the investigation of membrane proteins and transmembrane communication.

EPFL1997

Cell-type-specific nicotinic input disinhibits mouse barrel cortex during active sensing

Sylvain Crochet, Célia Roxane Gasselin, Benoît Hohl, Carl Petersen

Fast synaptic transmission relies upon the activation of ionotropic receptors by neurotransmitter release to evoke postsynaptic potentials. Glutamate and GABA play dominant roles in driving highly dynamic activity in synaptically connected neuronal circuits, but ionotropic receptors for other neurotransmitters are also expressed in the neocortex, including nicotinic receptors, which are non-selective cation channels gated by acetylcholine. To study the function of non-glutamatergic excitation in neocortex, we used two-photon microscopy to target whole-cell membrane potential recordings to different types of genetically defined neurons in layer 2/3 of primary somatosensory barrel cortex in awake head-restrained mice combined with pharmacological and optogenetic manipulations. Here, we report a prominent nicotinic input, which selectively depolarizes a subtype of GABAergic neuron expressing vasoactive intestinal peptide leading to disinhibition during active sensorimotor processing. Nicotinic disinhibition of somatosensory cortex during active sensing might contribute importantly to integration of top-down and motor-related signals necessary for tactile perception and learning.

CELL PRESS2021

Electrophysiology and fluorescence microscopy of ligand-gated ion channels

Christoph Schreiter

The thesis presented here describes studies of ligand binding, function, and arrangement of ligand-gated ion channels in cell membranes. Fluorescence microscopy and the patch clamp technique are used to investigate the nicotinic acetylcholine receptor and the serotonergic 5-HT3 receptor. Charge effects on ligand binding were investigated on the 5-HT3 receptor by site directed mutagenesis of strongly conserved charged amino acids close to the ligand-binding site. It turns out that steric effects have a higher impact on ligand binding than charges but that charged amino acids change the local environment strongly. This was exploited using a fluorescently labelled ligand to determine the distance of the bound ligand to the mutated amino acid. Ligand binding to the acetylcholine receptor was studied with a novel fluorescent ligand based on a toxin from the venom of marine snails (α-conotoxin GI).The fluorescent toxin acts as antagonist and can be bind completely reversible and with excellent specificity to muscle type acetylcholine receptors. Repetitive cycles of binding and unbinding were performed to obtain binding kinetics on single cells.The developed fluorescence microscope experiment enables the detection of only very low amount of fluorescent ligand from around 103 down to single molecules. The reversible binding was exploited to label single acetylcholine receptor and to monitor their diffusion in cell membranes. HEK293 cells served as a model system where co-expression of the anchoring protein rapsyn strongly influences receptor diffusion. Data were compared to diffusion in muscle cell lines during differentiation into myotubes. To obtain new information on ligand binding and channel gating, an experiment has been developed to simultaneously measure fluorescence and ion channel activity. The fluorescently labelled α-conotoxin GI was used to demonstrate the capability of this system. In addition, to increase the yield of electrophysiological measurements, a technique to perform patch clamp on a chip was further developed towards automation.

EPFL2005

Electrophysiology and fluorescence microscopy of ligand-gated ion channels

Christoph Schreiter

EPFL2005

Supported lipid monolayers for the investigation of specific interactions between proteins and ligands

EPFL1997