Calcium- and syntaxin 1-mediated trafficking of the neuronal glycine transporter GLYT2

Previously we demonstrated the existence of a physical and functional inter action between the glycine transporters and the SNARE protein syntaxin 1, I n the present report the physiological role of the syntaxin l-glycine trans porter 2 (GLYT2) interaction has been investigated by using a brain-derived preparation. Previous studies, focused on syntaxin 1-transporter interacti ons using overexpression systems, led to the postulation that syntaxin is s omehow implicated in protein trafficking. Since syntaxin 1 is involved in e xocytosis of neurotransmitter and also interacts with GLYT2, we stimulated exocytosis in synaptosomes and examined its effect on surface-expression an d transport activity of GLYT2, We found that, under conditions that stimula te vesicular glycine release, GLYT2 is rapidly trafficked first toward the plasma membrane and then internalized. When the same experiments were perfo rmed with synaptosomes inactivated for syntaxin 1 by a pretreatment with th e neurotoxin Bont/C, GLYT2 was unable to reach the plasma membrane but stil l was able to leave it. These results indicate the existence of a SNARE-med iated regulatory mechanism that controls the surface-expression of GLYT2. S yntaxin 1 is involved in the arrival to the plasma membrane but not in the retrieval. Furthermore, by using immunogold labeling on purified preparatio ns from synaptosomes, we demonstrate that GLYT2 is present in small synapti c-like vesicles. GLYT2-containing vesicles may represent neurotransmitter t ransporter that is being trafficked. The results of our work suggest a clos e correlation between exocytosis of neurotransmitter and its reuptake by tr ansporters.