Reversible sequential-binding probe receptor-ligand interactions in single cells.
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.
Schreiter
Christoph
C
Ecole Polytechnique Fédérale de Lausanne (EPFL), Laboratoire de Chimie Physique des Polymères et Membranes, 1015 Lausanne, Switzerland.
Gjoni
Marinela
M
Hovius
Ruud
R
Martinez
Karen L
KL
Segura
Jean-Manuel
JM
Vogel
Horst
H
D001667:Binding, Competitive; D020916:Conotoxins / Q000737:chemistry; D005456:Fluorescent Dyes; D007700:Kinetics; D008024:Ligands; D008904:Miniaturization; D018733:Nicotinic Antagonists / Q000737:chemistry; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry*; D011978:Receptors, Nicotinic / Q000737:chemistry; D014403:Tubocurarine / Q000737:chemistry
D020916:Conotoxins; D005456:Fluorescent Dyes; D008024:Ligands; D018733:Nicotinic Antagonists; D011956:Receptors, Cell Surface; D011978:Receptors, Nicotinic; D014403:Tubocurarine