Ligand-gated ion channel

Ligand-gated ion channels (LICs, LGIC), also commonly referred to as ionotropic receptors, are a group of transmembrane ion-channel proteins which open to allow ions such as Na+, K+, Ca2+, and/or Cl− to pass through the membrane in response to the binding of a chemical messenger (i.e. a ligand), such as a neurotransmitter. When a presynaptic neuron is excited, it releases a neurotransmitter from vesicles into the synaptic cleft. The neurotransmitter then binds to receptors located on the postsynaptic neuron. If these receptors are ligand-gated ion channels, a resulting conformational change opens the ion channels, which leads to a flow of ions across the cell membrane. This, in turn, results in either a depolarization, for an excitatory receptor response, or a hyperpolarization, for an inhibitory response. These receptor proteins are typically composed of at least two different domains: a transmembrane domain which includes the ion pore, and an extracellular domain which includes

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

In vivo protein labeling for structural and functional investigation of the 5-HT3A neurotransmitter receptor

Erwin Ilegems

In this thesis, two different fluorescent labeling techniques for in vivo investigations on the 5-HT3 receptor (5-HT3R) functions are presented. This plasma membrane protein contains five subunits surrounding an ion channel that opens after binding of a 5-HT3-specific neurotransmitter. The first technique, described in the first part of this report, focuses on receptor labeling via genetic fusion to spectral variants of the green fluorescent protein. I found that the resulting chimeras containing one fluorescent protein per subunit exhibit preserved ligand binding and channel activity, opening a wide range of biological research applications. Among these, I present the possibility to follow the 5-HT3R trafficking and localization during its entire life cycle by multicolor imaging in live cells, starting with its cytoplasmic biogenesis and ending with its ligand-induced internalization. The intracellular subunit assembly is shown to occur in the endoplasmic reticulum, and the importance of the cytoskeleton microtubules for proper membrane targeting is unraveled. The utility of bioluminescent 5-HT3R contructs was also demonstrated in another approach using the chemical disruption of cellular actin filaments to produce vesicular fractions of cells, in the order of 0.1 to a few micrometers in diameter. These so-called native vesicles, containing the labeled receptors in their membrane, were shown to be suitable for measurements using fluorescence confocal microscopy of ligand binding and of ion influx, opening new possibilities for miniaturized bioanalytics. In a third approach, I demonstrated that after detergent-solubilization of the receptor, the green fluorescent protein (GFP) inserted into the receptor sequence permitted to monitor ligand binding via fluorescence resonance energy transfer (FRET). Furthermore, I could observe a spatial reorientation of the receptor GFPs upon binding an agonist to the receptor. In the second part of this thesis, I adapted the mis-acylated suppressor tRNA technology to mammalian cells, permitting the introduction of unnatural amino acids at specific positions in the protein of interest. I achieved an efficiency of amino acid incorporation using in vitro aminoacylated suppressor tRNA in the order of 15% in CHO cells. A novel methodology for the quantification of background natural nonsense codon readthrough in different cell lines was also developed, permitting to select the most suitable codon-anticodon pair for this suppressor tRNA technique in various cell lines. Finally, I present a general strategy to increase the aforementioned artificial incorporation efficiency by down-regulating the competing eukaryotic release factor 1 (eRF1) using small interfering RNAs.

EPFL2004

In vivo protein labeling for structural and functional investigation of the 5-HT3A neurotransmitter receptor

Erwin Ilegems

EPFL2004

Electrophysiology and fluorescence microscopy of ligand-gated ion channels

Christoph Schreiter

EPFL2005

Mutation and analysis of a photoactivated ion channel. Channelrhodopsin from Volvox Carteri

Electrophysiology and fluorescence microscopy of ligand-gated ion channels

In vivo protein labeling for structural and functional investigation of the 5-HT3A neurotransmitter receptor

Mutation and analysis of a photoactivated ion channel. Channelrhodopsin from Volvox Carteri

In vivo protein labeling for structural and functional investigation of the 5-HT3A neurotransmitter receptor

Electrophysiology and fluorescence microscopy of ligand-gated ion channels