Formation of glycine receptor clusters and their accumulation at synapses

The glycine receptor is highly enriched in microdomains of the postsynaptic neuronal surface apposed to glycinergic afferent endings. There is substan tial evidence suggesting that the selective clustering of glycine receptor at these sites is mediated by the cytoplasmic protein gephyrin. To investig ate the formation of postsynaptic glycine receptor domains, we have examine d the surface insertion of epitope-tagged receptor alpha subunits in cultur ed spinal cord neurons after gene transfer by polyethylenimine-adenofection . Expression studies were also carried out using the non-neuronal cell line COS-7. Immunofluorescence microscopy was performed using wild-type isoform s and an alpha mutant subunit bearing the gephyrin-binding motif of the bet a subunit. In COS-7 cells, transfected glycine receptor alpha subunits had a diffuse surface distribution. Following cotransfection with gephyrin, onl y the mutant subunit formed cell surface clusters. In contrast, in neurons all subunits were able to form cell surface cluster s after transfection. These clusters were not colocalized with detectable e ndogenous gephyrin, and the GlyR beta subunit could not be detected in tran sfected cells. Therefore, exogenous receptors were not assembled as heterom eric complexes. A quantitative analysis demonstrated that newly synthesized glycine receptor progressively populated endogenous gephyrin clusters, sin ce association of both proteins increased as a function of time after the o nset of receptor synthesis. This phenomenon was accelerated when glycine re ceptor contained the gephyrin-binding domain. Together with previous results, these data support a two-step model for gly cinergic synaptogenesis whereby the gephyrin-independent formation of cell surface clusters precedes the gephyrin-mediated postsynaptic accumulation o f clusters.