title: A new function for glycine GlyT2 transporters: Stimulation of γ-aminobutyric acid release from cerebellar nerve terminals through GAT1 transporter reversal and Ca(2+)-dependent anion channels.
abstract: Glycine GlyT2 transporters are localized on glycine-storing nerve endings. Their main function is to accumulate glycine to replenish synaptic vesicles. Glycine was reported to be costored with γ-aminobutyric acid (GABA) in cerebellar interneurons that may coexpress glycine and GABA transporters, and this is confirmed here by confocal microscopy analysis showing coexpression of GAT1 and GlyT2 transporters on microtubule-associated protein-2-positive synaptosomes. It was found that GABA uptake elicited glycine release from cerebellar nerve endings by various mechanisms. We investigated whether and by what mechanisms activation of glycine transporters could mediate release of GABA. Nerve endings purified from cerebellum were prelabeled with [3H]GABA and exposed to glycine. Glycine stimulated [3H]GABA release in a concentration-dependent manner. The glycine effect was insensitive to strychnine or to 5,7-dichlorokynurenate but it was abolished when GlyT2 transporters were blocked. About 20% of the evoked release was dependent on external Ca2+ entered by reversal of plasmalemmal Na+/Ca2+ exchangers. A significant portion of the GlyT2-mediated release of [3H]GABA (about 50% of the external Ca(2+)-independent release) occurred by reversal of GABA GAT1 transporters. Na+ ions, reaching the cytosol during glycine uptake through GlyT2, activated mitochondrial Na+/Ca2+ exchangers, causing an increase in cytosolic Ca2+, which in turn triggered a Ca(2+)-induced Ca2+ release process at inositoltrisphosphate receptors. Finally, the increased availability of Ca2+ in the cytosol allowed the opening of anion channels permeable to GABA. In conclusion, GlyT2 transporters not only take up glycine to replenish synaptic vesicles but can also mediate release of GABA by reversal of GAT1 and permeation through anion channels.
authors.affiliation: Department of Pharmacy, Pharmacology and Toxicology Section, University of Genoa, Genoa, Italy; Center of Excellence for Biomedical Research, University of Genoa, Genoa, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Milanese
authors.forename: Cristina
authors.initials: C
authors.lastname: Romei
authors.forename: Cesare
authors.initials: C
authors.lastname: Usai
authors.forename: Martina
authors.initials: M
authors.lastname: Oliveri
authors.forename: Luca
authors.initials: L
authors.lastname: Raiteri
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002531:Cerebellum / Q000648:ultrastructure*; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018682:GABA Agents / Q000494:pharmacology; D050485:GABA Plasma Membrane Transport Proteins / Q000378:metabolism*; D005998:Glycine / Q000494:pharmacology; D018684:Glycine Agents / Q000494:pharmacology; D050487:Glycine Plasma Membrane Transport Proteins / Q000378:metabolism*; D007736:Kynurenic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D008297:Male; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009557:Nipecotic Acids / Q000494:pharmacology; D013574:Synaptosomes / Q000187:drug effects*; D014316:Tritium / Q000493:pharmacokinetics; D005680:gamma-Aminobutyric Acid / Q000493:pharmacokinetics
chemical_list: D002121:Calcium Channel Blockers; D018691:Excitatory Amino Acid Antagonists; D018682:GABA Agents; D050485:GABA Plasma Membrane Transport Proteins; D018684:Glycine Agents; D050487:Glycine Plasma Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D009557:Nipecotic Acids; C493270:Slc6a5 protein, mouse; D014316:Tritium; D005680:gamma-Aminobutyric Acid; C045598:N-(4,4-diphenyl-3-butenyl)nipecotic acid; D007736:Kynurenic Acid; D002118:Calcium; C066192:5,7-dichlorokynurenic acid; D005998:Glycine
keywords: GABA release; GAT1 GABA transporters; GlyT2 glycine transporters; IP3 receptors; Na+/Ca2+ exchangers; anion channels