Nerve terminals release neurotransmitters by exocytosis of synaptic vesicles, a process requiring prior accumulation within the vesicles. For “classical” (small-molecule) transmitters, this accumulation results from active transport of cytosolic transmitter across the vesicular membrane into the vesicle lumen. This transport involves a V-type proton-pumping ATPase, which acidifies and positively charges the vesicle lumen, and a secondary active transporter, which uses the proton electrochemical gradient across the vesicular membrane to accumulate specific transmitters (Edwards 2007).
The vesicular inhibitory amino acid transmitter (VIAAT), also known as vesicular GABA transporter (VGAT), ensures the vesicular uptake of the inhibitory amino acids GABA and glycine. VIAAT identification was initiated by genetic studies of GABAergic neurotransmission in the nematode worm Caenorhabditis elegans. Adult hermaphrodite worms have 26 GABAergic neurons, including inhibitory motoneurons involved in the reciprocal inhibition of opposite body wall muscles (McIntire et al. 1993a). Ablation of these motoneurons with a laser microbeam induced a specific motor behavior, which was used to identify mutant strains with defective GABAergic neurotransmission. Among the five selected genes analyzed further, unc-47 and unc-46 were implicated downstream from GABA synthesis but upstream of postsynaptic detection, suggesting that the corresponding proteins may be involved in vesicular GABA loading (McIntire et al. 1993b).
The unc-47 gene seemed a better candidate for this function because unc-47 mutant worms showed stronger GABA-related phenotypes and unc-47 neurons accumulated cytosolic GABA relative to the wild type, suggesting defective synaptic release (McIntire et al. 1993b). Two subsequent studies independently identified the UNC-47 protein using in vivo (mutant phenotype rescue) or in silico positional cloning approaches (McIntire et al. 1997; Sagné et al. 1997). A database search with the UNC-47 amino acid sequence in turn identified a single mammalian homologue, termed VIAAT or VGAT (McIntire et al. 1997; Sagné et al. 1997). The HUGO Gene Nomenclature Committee assigned the SLC32A1 symbol to the human gene, which appears to be the unique member of this family.
Transport Mechanism and Other Molecular Aspects
VIAAT belongs to the amino acid-polyamine-organocation (APC) superfamily of secondary active transporters (Saier 2000). The VIAAT polypeptide apparently folds into nine transmembrane helices with a large (∼100 amino acids) cytosolic N-terminal domain and a short C-terminus exposed to the vesicle lumen (Martens et al. 2008). The N terminus possesses an unconventional dileucine-like sorting motif involved in VIAAT recycling to synaptic vesicles after exocytosis (Santos et al. 2013).
In C. elegans, the intracellular trafficking of UNC-47 depends on UNC-46, a single-pass transmembrane protein showing homology with the lysosomal membrane proteins LAMP1 and LAMP2 (Schuske et al. 2007). This interaction explained the identification of unc-46 as a GABAergic gene and the similar phenotypes of unc-47 and unc-46 worms (McIntire et al. 1993b). However, UNC-46 does not seem to form an obligate heterodimer with UNC-47 since UNC-46-defective neurons showed residual GABA exocytosis, in contrast with UNC-47-defective neurons, and UNC-47 overexpression rescued the phenotype of unc-46 worms (Schuske et al. 2007). UNC-46 may instead operate as a chaperone assisting the sorting of UNC-47 to the cell bodies and varicosities of GABAergic neurons. A mammalian homologue of UNC-46, termed BAD-LAMP, has been identified (David et al. 2007). However, its role in the mammalian nervous system remains unknown.
The transport mechanism of VIAAT has been debated. Initial studies of GABA transport into synaptic vesicles showed a dependence on both pH and voltage vesicular gradients, suggesting that the transmitter accumulates through an exchange with luminal protons (Edwards 2007). More recently, a study of purified VIAAT reconstituted into proteoliposomes suggested instead a GABA/chloride cotransport mechanism (Juge et al. 2009). However, dynamic measurement of luminal pH and membrane voltage on single synaptic vesicles ruled out this mechanism and corroborated the GABA/H+ exchange mechanism, with no major contribution of Cl−, K+, or Na+ (Farsi et al. 2016). Moreover, GABA/H+ exchange is consistent with the slightly higher pH of GABAergic vesicles relative to glutamatergic vesicles in live neurons and with the biphasic pH dynamics of recycling GABAergic vesicles; after an initial phase of fast reacidification, the luminal pH increases with ∼20-s time constant at physiological temperature, presumably reflecting slow refilling with GABA in exchange for protons (Egashira et al. 2016).
Role in GABAergic and Glycinergic Neurotransmission
VIAAT is expressed in both GABAergic and glycinergic neurons (Chaudhry et al. 1998; Dumoulin et al. 1999; Sagné et al. 1997) and its genetic inactivation in mouse abolished or drastically reduced the amplitude of both GABAergic and glycinergic postsynaptic currents (Wojcik et al. 2006). A single transporter is thus responsible for the vesicular loading of both amino acids, thus accounting for the corelease of GABA and glycine at some central synapses (Jonas 1998).
It should be noted, however, that the two inhibitory transmitters have distinct requirements for efficient vesicular loading, presumably because of the lower affinity for glycine relative to GABA. Vesicular loading, and release, of GABA critically depends on bulk transmitter synthesis by the glutamic acid decarboxylase isoform GAD67 in GABAergic neurons. However, its dependence on local GABA synthesis by the vesicle-attached GAD65 isoform is more subtle, with a significant effect of GAD65 inactivation observed only during sustained synaptic activation (Tian et al. 1999). In contrast, the level of cytosolic glycine achieved by biosynthesis does not allow significant vesicular loading and local cytosolic accumulation by the concentrative plasma membrane transporter GlyT2 is required to achieve significant vesicular loading (Aubrey et al. 2007). This cooperation between VIAAT and GlyT2 underlies the impairment of glycinergic transmission in hyperekplexia patients with GlyT2 mutations (Rees et al. 2006).
Nerve terminals release small-molecule neurotransmitters by exocytosis of synaptic vesicles, a process requiring prior accumulation within the vesicle lumen following uptake by a specific transporter. The vesicular inhibitory amino acid transmitter (VIAAT), also known as VGAT, ensures this accumulation for GABA and glycine at inhibitory nerve terminals. The existence of a shared transporter accounts for the corelease of the two inhibitory transmitters at some central synapses. However, vesicular loading of the two transmitters show distinct dependence on cytosolic biosynthesis and plasma membrane uptake presumably because of the very low affinity of VIAAT for glycine.