title: Endocytosis of the neuronal glycine transporter GLYT2: role of membrane rafts and protein kinase C-dependent ubiquitination.
abstract: Glycinergic neurotransmission is terminated by sodium- and chloride-dependent plasma membrane transporters. The neuronal glycine transporter 2 (GLYT2) supplies the terminal with substrate to refill synaptic vesicles containing glycine. This crucial process is defective in human hyperekplexia, a condition that can be caused by mutations in GLYT2. Inhibitory glycinergic neurotransmission is modulated by the GLYT2 exocytosis/endocytosis equilibrium, although the mechanisms underlying the turnover of this transporter remain elusive. We studied GLYT2 internalization pathways and the role of ubiquitination and membrane raft association of the transporter in its endocytosis. Using pharmacological tools, dominant-negative mutants and small-interfering RNAs, we show that the clathrin-mediated pathway is the primary mechanism for constitutive and regulated GLYT2 endocytosis in heterologous cells and neurons. We show that GLYT2 is constitutively internalized from cell surface lipid rafts, remaining associated with rafts in subcellular recycling structures. Protein kinase C (PKC) negatively modulates GLYT2 via rapid and dynamic redistribution of GLYT2 from raft to non-raft membrane subdomains and increasing ubiquitinated GLYT2 endocytosis. This biphasic mechanism is a versatile means to modulate GLYT2 behavior and hence, inhibitory glycinergic neurotransmission. These findings may reveal new therapeutic targets to address glycinergic pathologies associated with alterations in GLYT2 trafficking.
authors.affiliation: Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Jaime
authors.initials: J
authors.lastname: de Juan-Sanz
authors.forename: Francisco
authors.initials: F
authors.lastname: Zafra
authors.forename: Beatriz
authors.initials: B
authors.lastname: López-Corcuera
authors.forename: Carmen
authors.initials: C
authors.lastname: Aragón
mesh_terms: D000818:Animals; D019556:COS Cells; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism; D004285:Dogs; D034285:Dynamin II / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D005089:Exocytosis / Q000502:physiology; D050487:Glycine Plasma Membrane Transport Proteins / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D009435:Synaptic Transmission / Q000502:physiology; D013572:Synaptic Vesicles / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology*
chemical_list: D002966:Clathrin; D050487:Glycine Plasma Membrane Transport Proteins; D008563:Membrane Lipids; D008565:Membrane Proteins; C493269:Slc6a5 protein, rat; D011493:Protein Kinase C; D034285:Dynamin II