Dynamics of glycine receptor insertion in the neuronal plasma membrane

The exocytosis site of newly synthesized glycine receptor was defined by me ans of a morphological assay to characterize its export from the trans-Golg i Network to the plasma membrane. This was achieved by expressing in transf ected neurons an alpha1 subunit bearing an N-terminal tag selectively cleav able from outside the cell by thrombin. This was combined with a transient temperature-induced block of exocytic transport that creates a synchronized exocytic wave. Immunofluorescence microscopy analysis of the cell surface appearance of newly synthesized receptor revealed that exocytosis mainly oc curred at nonsynaptic sites in the cell body and the initial portion of den drites. At the time of cell surface insertion, the receptors existed as dis crete clusters. Quantitative analysis showed that glycine receptor clusters are stable in size and subsequently appeared in more distal dendritic regi ons. This localization resulted from diffusion in the plasma membrane and n ot from exocytosis of transport vesicles directed to dendrites. Kinetic ana lysis established a direct substrate-product relationship between pools of somatic and dendritic receptors. This indicated that clusters represent int ermediates between newly synthesized and synaptic receptors. These results support a diffusion-retention model for the formation of receptor-enriched postsynaptic domains and not that of a vectorial intracellular targeting to synapses.