Regulation of glycine transporters

The regulation of neurotransmitter transporters is a central aspect of thei r physiology. Recent studies that focused on syntaxin-1 transporter interac tions led to the postulation that syntaxin-1 is somehow implicated in prote in trafficking. Because syntaxin-1 is involved in the exocytosis of neurotr ansmitters and it interacts with glycine transporter 2 (GLYT2), we stimulat ed exocytosis in synaptosomes and examined its effect on GLYT2 surface-expr ession and transport activity. We found that GLYT2 is rapidly trafficked fi rst towards the plasma membrane and then internalized under conditions that stimulate vesicular glycine release. However, when syntaxin-1 was inactiva ted by pre-treatment of synaptosomes with the botulinum neurotoxin C, GLYT2 was unable to reach the plasma membrane but still was able to leave it. Th ese results indicate the existence of a SNARE (soluble N-ethylmaleimide-sen sitive factor attachment protein receptor)mediated regulatory mechanism tha t controls the surface expression of GLYT2. Syntaxin-1 is involved in the t ransport of GLYT2 to, but not its retrieval from, the plasma membrane. Immu nogold-labelling on purified vesicular preparations from synaptosomes showe d that GLYT2 is present in sm. all synaptic-like vesicles. This may represe nt neurotransmitter transporter that is being trafficked. The subcellular d istribution of the glycine transporters was further examined in PC12 cells that were stably transfected with the fusions of GLYT1 and GLYT2 with green fluorescent protein. There was a clear difference in their intracellular d istribution, GLYT1 being present mainly on the plasma membrane and GLYT2 be ing localized mainly on large, dense-core vesicles. We are trying to find s ignal sequences responsible for this differential localization.