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Multiple Association States between Glycine Receptors and Gephyrin Identified by SPT Analysis

Marie-Virginie Ehrensperger ^*, Cyril Hanus , Christian Vannier , Antoine Triller  and Maxime Dahan ^*

^* Laboratoire Kastler Brossel, Centre National de la Recherche Scientifique UMR8552, Ecole normale supérieure, Université Pierre et Marie Curie-Paris 6, 75005 Paris, France; and Biologie Cellulaire de la Synapse, Institut National de la Sante et de la Recherche Medicale U789, Ecole normale supérieure, 75005 Paris, France

Correspondence: Address reprint requests to Maxime Dahan, E-mail: maxime.dahan{at}lkb.ens.fr.

The scaffolding protein gephyrin is known to anchor glycine receptors (GlyR) at synapses and to participate in the dynamic equilibrium between synaptic and extrasynaptic GlyR in the neuronal membrane. Here we investigated the properties of this interaction in cells cotransfected with YFP-tagged gephyrin and GlyR subunits possessing an extracellular myc-tag. In HeLa cells and young neurons, single particle tracking was used to follow in real time individual GlyR, labeled with quantum dots, traveling into and out of gephyrin clusters. Analysis of the diffusion properties of two GlyR subunit types—able or unable to bind gephyrin—gave access to the association states of GlyR with its scaffolding protein. Our results indicated that an important portion of GlyR could be linked to a few molecules of gephyrin outside gephyrin clusters. This emphasizes the role of scaffolding proteins in the extrasynaptic membrane and supports the implication of gephyrin-gephyrin interactions in the stabilization of GlyR at synapses. The kinetic parameters controlling the equilibrium between GlyR inside and outside clusters were also characterized. Within clusters, we identified two subpopulations of GlyR with distinct degrees of stabilization between receptors and scaffolding proteins.