title: GABA transporters mediate glycine release from cerebellum nerve endings: roles of Ca(2+)channels, mitochondrial Na(+)/Ca(2+) exchangers, vesicular GABA/glycine transporters and anion channels.
abstract: GABA transporters accumulate GABA to inactivate or reutilize it. Transporter-mediated GABA release can also occur. Recent findings indicate that GABA transporters can perform additional functions. We investigated how activation of GABA transporters can mediate release of glycine. Nerve endings purified from mouse cerebellum were prelabeled with [(3)H]glycine in presence of the glycine GlyT1 transporter inhibitor NFPS to label selectively GlyT2-bearing terminals. GABA was added under superfusion conditions and the mechanisms of the GABA-evoked [(3)H]glycine release were characterized. GABA stimulated [(3)H]glycine release in a concentration-dependent manner (EC(50) = 8.26 μM). The GABA-evoked release was insensitive to GABA(A) and GABA(B) receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca(2+) entering the nerve terminals through VSCCs sensitive to ω-conotoxins. The external Ca(2+)-independent release involved mitochondrial Ca(2+), as it was prevented by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca(2+) did not trigger exocytotic release because the [(3)H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [(3)H]glycine so that less [(3)H]glycine can become cytosolic when GABA taken up exchanges with [(3)H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [(3)H]glycine efflux could be prevented by niflumic acid or NPPB indicating that the evoked release occurred essentially by permeation through anion channels. In conclusion, GABA uptake into GlyT2-bearing cerebellar nerve endings triggered glycine release which occurred essentially by permeation through Ca(2+)-dependent anion channels. Glial GABA release mediated by anion channels was proposed to underlie tonic inhibition in the cerebellum; the present results suggest that glycine release by neuronal anion channels also might contribute to tonic inhibition.
authors.affiliation: Department of Experimental Medicine, Section of Pharmacology and Toxicology, University of Genoa, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Romei
authors.forename: Maurizio
authors.initials: M
authors.lastname: Raiteri
authors.forename: Luca
authors.initials: L
authors.lastname: Raiteri
mesh_terms: D000818:Animals; D000838:Anions / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018756:GABA Antagonists / Q000494:pharmacology; D050485:GABA Plasma Membrane Transport Proteins / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D005998:Glycine / Q000378:metabolism*; D050487:Glycine Plasma Membrane Transport Proteins / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism; D008297:Male; D051379:Mice; D009411:Nerve Endings / Q000187:drug effects / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D050496:Vesicular Glutamate Transport Proteins / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D000838:Anions; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D018756:GABA Antagonists; D050485:GABA Plasma Membrane Transport Proteins; D050487:Glycine Plasma Membrane Transport Proteins; D007473:Ion Channels; D019831:Sodium-Calcium Exchanger; D050496:Vesicular Glutamate Transport Proteins; D005680:gamma-Aminobutyric Acid; D002118:Calcium; D005998:Glycine