The glycine transporter GlyT2 controls the dynamics of synaptic vesicle refilling in inhibitory spinal cord neurons.
At inhibitory synapses, glycine and GABA are accumulated into synaptic vesicles by the same vesicular transporter VGAT/VIAAT (vesicular GABA transporter/vesicular inhibitory amino acid transporter), enabling a continuum of glycine, GABA, and mixed phenotypes. Many fundamental aspects of the presynaptic contribution to the inhibitory phenotypes remain unclear. The neuronal transporter GlyT2 is one of the critical presynaptic factors, because glycinergic transmission is impaired in knock-out GlyT2(-/-) mice and mutations in the human GlyT2 gene slc6a5 are sufficient to cause hyperekplexia. Here, we establish that GlyT2-mediated uptake is directly coupled to the accumulation of glycine into recycling synaptic vesicles using cultured spinal cord neurons derived from GlyT2-enhanced green fluorescent protein transgenic mice. Membrane expression of GlyT2 was confirmed by recording glycine-evoked transporter current. We show that GlyT2 inhibition induces a switch from a predominantly glycine to a predominantly GABA phenotype. This effect was mediated by a reduction of glycinergic quantal size after cytosolic depletion of glycine and was entirely reversed by glycine resupply, illustrating that the filling of empty synaptic vesicles is tightly coupled to GlyT2-mediated uptake. Interestingly, high-frequency trains of stimuli elicit two phases of vesicle release with distinct kinetic requirements for glycine refilling. Thus, our results demonstrate the central role played by GlyT2 in determining inhibitory phenotype and therefore in the physiology and pathology of inhibitory circuits.
Rousseau
France
F
Laboratoire de Neurobiologie, Ecole Normale Supérieure, 75005 Paris, France.
Aubrey
Karin R
KR
Supplisson
Stéphane
S
D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D018929:Cell Culture Techniques; D004307:Dose-Response Relationship, Radiation; D004347:Drug Interactions; D004558:Electric Stimulation / Q000379:methods; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D005998:Glycine / Q000378:metabolism / Q000494:pharmacology; D018684:Glycine Agents / Q000494:pharmacology; D050487:Glycine Plasma Membrane Transport Proteins / Q000235:genetics / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D053444:Inhibitory Postsynaptic Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009433:Neural Inhibition / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D017711:Nonlinear Dynamics; D018408:Patch-Clamp Techniques / Q000379:methods; D011724:Pyridazines / Q000494:pharmacology; D013116:Spinal Cord / Q000166:cytology*; D013331:Strychnine / Q000494:pharmacology; D013572:Synaptic Vesicles / Q000378:metabolism*; D013997:Time Factors
C532849:4-benzyloxy-3,5-dimethoxy-N-(1-(dimethylaminocyclopently)methyl)benzamide; D001549:Benzamides; D018691:Excitatory Amino Acid Antagonists; D018756:GABA Antagonists; D018684:Glycine Agents; D050487:Glycine Plasma Membrane Transport Proteins; D011724:Pyridazines; C493270:Slc6a5 protein, mouse; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C049853:gabazine; D013331:Strychnine; D005998:Glycine