Multiple association states between glycine receptors and
gephyrin identified by SPT analysis
Marie-Virginie Ehrensperger 1, Cyril Hanus 2, Christian Vannier 2, Antoine Triller 2 and Maxime Dahan 1*
1 Laboratoire Kastler Brossel, CNRS UMR8552; Ecole Normal Supérieure; Université Paris 6
2 Laboratoire de Biologie Cellulaire de la Synapse, INSERM U789 ; Ecole Normale Supérieure
* To whom correspondence should be addressed. E-mail: maxime.dahan{at}lkb.ens.fr.
Submitted on August 22, 2006
Revised on October 18, 2006
Accepted on 11 January 2007
 |
Abstract |
|---|
The scaffolding protein gephyrin is known to anchor glycine receptors (GlyR) at synapses and to participate to 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, labelled with quantum dots, travelling in 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.
Key Words:
Dynamic equilibrium, Glycine receptor and gephyrin, Lateral Diffusion, Quantum dots, Single Particle Tracking, Spinal cord neuron
