Acetylcholine receptor | EPFL Graph Search

An acetylcholine receptor (abbreviated AChR) is an integral membrane protein that responds to the binding of acetylcholine, a neurotransmitter. Classification Like other transmembrane receptors, acetylcholine receptors are classified according to their "pharmacology," or according to their relative affinities and sensitivities to different molecules. Although all acetylcholine receptors, by definition, respond to acetylcholine, they respond to other molecules as well. *Nicotinic acetylcholine receptors (nAChR, also known as "ionotropic" acetylcholine receptors) are particularly responsive to nicotine. The nicotine ACh receptor is also a Na+, K+ and Ca2+ ion channel. *Muscarinic acetylcholine receptors (mAChR, also known as "metabotropic" acetylcholine receptors) are particularly responsive to muscarine. Nicotinic and muscarinic are two main kinds of "cholinergic" receptors. Receptor types Molecular biology has shown that the nicotinic and muscarinic receptors

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

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

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

Matteo Costioli, Rudolf Hovius, Horst Pick, Christoph Schreiter, Horst Vogel

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 mutagenesis. Wild type and mutant receptor proteins were expressed in HEK293 cells and analyzed by electrophysiol., radioligand binding, fluorescence measurements, and immunochem. A structural model of the ligand-binding domain of the 5-HT3R based on the acetylcholine binding protein revealed the position of the mutated amino acids. Our results demonstrate that mutations of Glu97, distant from the ligand-binding site, had little effect on the receptor, whereas mutations Glu224 and Glu235, close to the predicted binding site, are indeed important for ligand binding. Mutations E224Q, E224D, and E235Q decreased EC50 and Kd values 5-20-fold, whereas E235D was functionally expressed at a low level and had a more than 100-fold increased EC50 value. Comparison of the fluorescence properties of a fluorescein-labeled antagonist upon binding to wild type 5-HT3R and E235Q, allowed us to localize Glu235 within a distance of 1 nm around the ligand-binding site, as proposed by our model. [on SciFinder (R)]

2003

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

EPFL1997

Electrophysiology and fluorescence microscopy of ligand-gated ion channels

Christoph Schreiter

EPFL2005