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-outGlyT2^−/−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.

The mechanisms that specify the vesicular phenotype of inhibitory interneurons in vertebrates are poorly understood because the two main inhibitory transmitters, glycine and GABA, share the same vesicular inhibitory amino acid transporter (VIAAT) and are both present in neurons during postnatal development. We have expressed VIAAT and the plasmalemmal transporters for glycine and GABA in a neuroendocrine cell line and measured the quantal release of glycine and GABA using a novel double-sniffer patch-clamp technique. We found that glycine is released from vesicles when VIAAT is coexpressed with either the neuronal transporter GlyT2 or the glial transporter GlyT1. However, GlyT2 was more effective than GlyT1, probably because GlyT2 is unable to operate in the reverse mode, which gives it an advantage in maintaining the high cytosolic glycine concentration required for efficient vesicular loading by VIAAT. The vesicular inhibitory phenotype was gradually altered from glycinergic to GABAergic through mixed events when GABA is introduced into the secretory cell and competes for uptake by VIAAT. Interestingly, the VIAAT ortholog from Caenorhabditis elegans (UNC-47), a species lacking glycine transmission, also supports glycine exocytosis in the presence of GlyT2, and a point mutation of UNC-47 that abolishes GABA transmission in the worm confers glycine specificity. Together, these results suggest that an increased cytosolic availability of glycine in VIAAT-containing terminals was crucial for the emergence of glycinergic transmission in vertebrates.

N‐arachidonyl‐glycine is one of a series of N‐arachidonyl‐amino acids that are derived from arachidonic acid. N‐arachidonyl‐glycine is produced in a wide range of tissues with greatest abundance in the spinal cord. Here we report that N‐arachidonyl‐glycine is a reversible and non‐competitive inhibitor of glycine transport by GLYT2a, but has little effect on glycine transport by GLYT1b or γ‐amino butyric acid transport by GAT1. It has previously been reported that the activity of GLYT2a is down‐regulated by protein kinase C and therefore we investigated whether the actions of N‐arachidonyl‐glycine on GLYT2a are mediated by second messenger systems that lead to the activation of protein kinase C. However, the protein kinase C inhibitor, staurosporine, had no effect on the actions of N‐arachidonyl‐glycine on GLYT2a. Thus, the actions of N‐arachidonyl‐glycine are likely to be mediated by a direct interaction with the transporter. We have further defined the pharmacophore by investigating the actions of other N‐arachidonyl amino acids as well as the closely related compounds arachidonic acid, anandamide and R1‐methanandamide. Arachidonic acid, anandamide and R1‐methanandamide have no effect on glycine transport, but N‐arachidonyl‐l‐alanine has similar efficacy at GLYT2a to N‐arachidonyl‐glycine, and N‐arachidonyl‐γ‐amino butyric acid is less efficacious. These observations define a novel recognition site for the N‐arachidonyl amino acids.