Reversible sequential-binding probe receptor-ligand interactions in single cells |
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Authors: | Schreiter Christoph Gjoni Marinela Hovius Ruud Martinez Karen L Segura Jean-Manuel Vogel Horst |
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Affiliation: | Ecole Polytechnique Fédérale de Lausanne (EPFL), Laboratoire de Chimie Physique des Polymères et Membranes, 1015 Lausanne, Switzerland. |
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Abstract: | With the reversible sequential (ReSeq) binding assay,we present a novel approach for the ultrasensitive profiling of receptor function in single living cells. This assay is based on the repetitive application of fluorescent ligands that have fast association-dissociation kinetics. We chose the nicotinic-acetylcholine receptor (nAChR) as a prototypical example and performed ReSeq equilibrium, kinetic, and competition-binding assays using fluorescent derivatives of the antagonist alpha-conotoxin GI (alpha-CnTx). Thereby, we determined the binding constants of unlabeled alpha-CnTx and d-tubocurarine. The high selectivity of alpha-CnTx for muscle-type nAChR made it possible to observe specific binding even in the presence of other nAChR subtypes. Imaging of individual nAChRs and ligand-binding cycles to single cells in microfluidic devices demonstrated the ultimate miniaturization and accuracy of ReSeq-binding assays even at low receptor-expression levels. We expect our approach to be of generic importance for functional screening of compounds or membrane receptors, and for the detailed characterization of rare primary cells. |
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Keywords: | binding assays fluorescent probes ion channels nicotinic‐acetylcholine receptor single‐molecule studies |
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