Calcium- and syntaxin 1-mediated trafficking of the neuronal glycine transporter GLYT2.
Previously we demonstrated the existence of a physical and functional interaction between the glycine transporters and the SNARE protein syntaxin 1. In the present report the physiological role of the syntaxin 1-glycine transporter 2 (GLYT2) interaction has been investigated by using a brain-derived preparation. Previous studies, focused on syntaxin 1-transporter interactions using overexpression systems, led to the postulation that syntaxin is somehow implicated in protein trafficking. Since syntaxin 1 is involved in exocytosis of neurotransmitter and also interacts with GLYT2, we stimulated exocytosis in synaptosomes and examined its effect on surface-expression and transport activity of GLYT2. We found that, under conditions that stimulate vesicular glycine release, GLYT2 is rapidly trafficked first toward the plasma membrane and then internalized. When the same experiments were performed with synaptosomes inactivated for syntaxin 1 by a pretreatment with the neurotoxin Bont/C, GLYT2 was unable to reach the plasma membrane but still was able to leave it. These results indicate the existence of a SNARE-mediated regulatory mechanism that controls the surface-expression of GLYT2. Syntaxin 1 is involved in the arrival to the plasma membrane but not in the retrieval. Furthermore, by using immunogold labeling on purified preparations from synaptosomes, we demonstrate that GLYT2 is present in small synaptic-like vesicles. GLYT2-containing vesicles may represent neurotransmitter transporter that is being trafficked. The results of our work suggest a close correlation between exocytosis of neurotransmitter and its reuptake by transporters.
Centro de Biologia Molecular Severo Ochoa, Facultad de Ciencias, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Cientificas, 28049 Madrid, Spain.
A
A
Geerlings
E
E
Núñez
B
B
López-Corcuera
C
C
Aragón
D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D001905:Botulinum Toxins / Q000494:pharmacology; D019274:Botulinum Toxins, Type A; D001933:Brain Stem / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D050487:Glycine Plasma Membrane Transport Proteins; D007700:Kinetics; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D013116:Spinal Cord / Q000378:metabolism; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D050827:Syntaxin 1
D026921:Amino Acid Transport Systems, Neutral; D000954:Antigens, Surface; D002352:Carrier Proteins; D050487:Glycine Plasma Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493269:Slc6a5 protein, rat; C494213:Stx1a protein, rat; D050827:Syntaxin 1; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D002118:Calcium; D005998:Glycine