FLUOROPATCH

Direct observation of ligand binding and channel gating on individual ligand-gated ion channels

Coordinatore	Universitätsklinikum Jena    more  Organization address city: Jena postcode: 7740 contact info Titolo: Ms. Nome: Nicole Cognome: Baier Email: send email Telefono: +49 3641 934350 Fax: +49 3641 933202
Nazionalità Coordinatore	Germany [DE]
Totale costo	45˙000 €
EC contributo	45˙000 €
Programma	FP7-PEOPLE Specific programme "People" implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013)
Code Call	FP7-PEOPLE-2009-RG
Funding Scheme	MC-ERG
Anno di inizio	2010
Periodo (anno-mese-giorno)	2010-04-01   -   2013-06-30

 Partecipanti

#	participant	country	role	EC contrib. [€]
1	Nome Ente NON disponibile  Organization address city: Jena postcode: 7740 contact info Titolo: Ms. Nome: Nicole Cognome: Baier Email: send email Telefono: +49 3641 934350 Fax: +49 3641 933202	DE (Jena)	coordinator	45˙000.00

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 Obiettivo del progetto (Objective)

'Understanding the dynamics of ligand-gated ion channels is essential for understanding synaptic processes and neuronal signal integration (neurotransmitter gated channels) and intracellular signalling (cyclic nucleotide gated channels). Here we will combine single molecule fluorescence detection, with single channel electrophysiology, to detect ligand-binding and follow subsequent channel gating simultaneously. Single-channel patch-clamp alone can only measure two experimental states and their development in time: open and closed. Still, given some assumption, such data permitted construction of detailed kinetic models. Fluorescence detection can determines whether ligands are present or not, and the kinetics of the binding process. However, the nature of the ligand (agonist or antagonist) is not directly accessible. It was suggested more than 10 years [Edelstein 1997], that combining the two approaches on the single-molecule level will gain insights into the receptor mechanism. We will combine the two approaches to directly link the two molecular functional determining events (ligand binding and channel gating) within one experiment. Recently we implemented a combination of confocal single molecule fluorescence detection with single channel patch clamp, and showed (on the example of nAChR and fluorescent epibatidine) that such experiments are technically possible and feasible [manuscript in preparation]. We identified two parameters as limiting for the achievable time resolutions: counting noise and ligand diffusion into the confocal volume. Scope of this project includes: (1) identifying and analyzing biological systems with kinetics accessible to this approach, (2) developing techniques to reduce the time limitation, in particular the use of FRET to avoid the diffusion delay and (3) propose a model to describe the intra-molecular single transduction, and discuss it in comparison to models derived from single channel patch clamp alone.'