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Direct observation of ligand binding and channel gating on individual ligand gat...

Direct observation of ligand binding and channel gating on individual ligand gated ion channels 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 chan... ver más

30/06/2013

UNIVERSITATSKLINIK...

45K€

Presupuesto del proyecto: 45K€

Líder del proyecto

UNIVERSITATSKLINIKUM JENA No se ha especificado una descripción o un objeto social para esta compañía.

TRL 4-5

Fecha límite participación Sin fecha límite de participación.

Financiación concedida El organismo FP7 notifico la concesión del proyecto el día 2013-06-30 No tenemos la información de la convocatoria

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UNIVERSITATSKLINIKUM JENA

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Líder del proyecto

UNIVERSITATSKLINIKUM JENA No se ha especificado una descripción o un objeto social para esta compañía.

TRL 4-5

Presupuesto del proyecto 45K€

Fecha límite de participación Sin fecha límite de participación.

Descripción del proyecto 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.

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