The Transporters GlyT2 and VIAAT Cooperate to Determine the Vesicular Glycinergic Phenotype

Abstract

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.

Distinct Neurochemical and Functional Properties of GAD67-Containing 5-HT Neurons in the Rat Dorsal Raphe Nucleus

“…At conventional synapses, GABA is released into the synaptic cleft by exocytosis of GABA-filled vesicles. In the present study, expression of VIAAT, which fills GABA and glycine into synaptic vesicles (McIntire et al, 1997;Aubrey et al, 2007), was not detected in 5-HT/GAD67 neurons by single-cell RT-PCR, double FISH, and immunofluorescence. This unexpected finding prompted us to hypothesize that 5-HT/GAD67 neurons might release GABA into the extracellular space via nonvesicular release.…”

Glycinergic transmission: glycine transporter GlyT2 in neuronal pathologies

“…However, it is unclear if this represents the third Na + -binding site that energizes the transport of glycine. In order to become glycinergic, a neuron not only requires GlyT2 on its surface but also a vesicular glycine transporter in the synaptic boutons [34]. This transporter, termed VIAAT or VGAT, belongs to the SLC32 family and uses the electrochemical proton gradient generated in synaptic vesicles by the H + -ATPase to accumulate glycine into the vesicular lumen [35].…”

Glycine transport inhibitors for the treatment of pain

“…First, the transporter is able to reduce the synaptic glycine concentration to the low nM range, under equilibrium conditions, to prevent low level GlyR activity, and second, the extra concentrating capacity allows the transporter to maintain the intracellular glycine concentration at approximately 10-20 mM, which is necessary to provide sufficient glycine for transport into synaptic vesicles via the vesicular inhibitory amino acid transporter (VIAAT) [30] (Box 1). VIAAT is non-selective for glycine over GABA and it will accumulate either neurotransmitter which is partly dependent on the neurotransmitter concentration in the presynaptic terminal [31]. Thus, for a glycinergic neuron it is important for the intracellular glycine concentration to be maintained at levels that allow glycine loading of vesicles [31].…”

“…VIAAT is non-selective for glycine over GABA and it will accumulate either neurotransmitter which is partly dependent on the neurotransmitter concentration in the presynaptic terminal [31]. Thus, for a glycinergic neuron it is important for the intracellular glycine concentration to be maintained at levels that allow glycine loading of vesicles [31]. In interneurones of the spinal cord, both glycine and GABA can be present in the same synaptic vesicles, which is presumably due to the presence of both GlyT2 and a GABA transporter capable of maintaining the intracellular GABA concentration at similar levels as glycine [31,32].…”

“…Thus, for a glycinergic neuron it is important for the intracellular glycine concentration to be maintained at levels that allow glycine loading of vesicles [31]. In interneurones of the spinal cord, both glycine and GABA can be present in the same synaptic vesicles, which is presumably due to the presence of both GlyT2 and a GABA transporter capable of maintaining the intracellular GABA concentration at similar levels as glycine [31,32]. However, segregation of GABAergic and glycinergic neurotransmission in dorsal spinal cord is extensive [11,19,28], with GABAergic synapses dominating in laminae I and IIo, and glycinergic transmission dominating in laminae Iii and III.…”