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Structural dynamics and cellular distribution of ligand-gated ion channels: Single molecule studies

English title Structural dynamics and cellular distribution of ligand-gated ion channels: Single molecule studies
Applicant Vogel Horst
Number 109886
Funding scheme Project funding (Div. I-III)
Research institution Laboratoire de Chimie Physique des Polymères et Membranes EPFL - SB - ISIC - LCPPM
Institution of higher education EPF Lausanne - EPFL
Main discipline Biophysics
Start/End 01.10.2005 - 31.12.2007
Approved amount 251'300.00
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All Disciplines (3)

Discipline
Biophysics
Biochemistry
Cellular Biology, Cytology

Keywords (8)

Structural dynamics of ion channels; biogenesis & trafficking of neuroreceptors; synapse formation; electrophysiology; single molecule fluorescence spectroscopy; & imaging

Lay Summary (English)

Lead
Lay summary
Ligand-gated ion channels (LGICs) play a central role in the rapid synaptic signal transduction. The trans-membrane proteins combine a receptor function, the sensing of ligands in the extracellular fluids, with an effector function, the subsequent opening of an intrinsic ion channel, resulting in activation or inhibition of a post-synaptic cell.Despite their importance for functioning of many biological cells and the fact that they are major targets of presently used medicines, central questions about the molecular architecture and the cellular function of LGIC remain to be solved.Here, we plan to continue to investigate two representative LGICs, the muscle-type nicotinic acetylcholine receptor (nAchR) and the serotonin5HT3 receptor (5HT3R), and will focus on the following:How does a ligand bind and induce gating of an ion channel?The central question how binding of an agonist on the extracellular side of a LGIC induces transmembrane structural changes in the protein, which finally results in opening of the ion channel is unresolved. Here we will investigate the structural dynamics of these transmembrane signallingevents: i) The orientation of agonists in the ligand binding site of nAchR will be determined using agonists comprising groups reactive to single Cys residue mutant receptors. ii) Conformational changes of nAchR and 5HT3R in both the ligand binding and channel domain will be investigated by the chemical accessibility of engineered single Cys-residues, by changes in fluorescence signals of fluorescent probes introduced in respective domains, and by parallel channel conductance measurements. iii) Measuring simultaneously the electrical ion conductance and the fluorescence of a single LGIC using fluorescent agonists and fluorescent receptors we aim to resolve how structural fluctuations between different receptor (sub)states finally open a channel.How does a cell traffick LGICs and how does this affect cellular signalling?Presence and activity of LGICs on the cell surface is highly regulated.In-vivo fluoresce labelling will be used to study biogenesis, plasma membrane targeting, internalisation and recycling of LGIC under various conditions like prolonged agonist exposure. Moreover, the distribution of LGICs on the cell surface plays an important role in synapse formation.Fluorescence imaging will be applied to study diffusion and lateral distribution of single receptor molecules during the development of muscle cells and of the neuromuscular junction. The role of so-called scaffold proteins will be addressed.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

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Associated projects

Number Title Start Funding scheme
118148 Structural dynamics and cellular distribution of ligand-gated ion channels: single molecule studies 01.01.2008 Project funding (Div. I-III)
102062 Structure and dynamic of ionotropic receptors 01.10.2003 Project funding (Div. I-III)

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