Vesicular Inhibitory Amino Acid Transporter Is a Cl−/γ-Aminobutyrate Co-transporter

Abstract

The vesicular inhibitory amino acid transporter (VIAAT) is a synaptic vesicle protein responsible for the vesicular storage of ␥-aminobutyrate (GABA) and glycine which plays an essential role in GABAergic and glycinergic neurotransmission. The transport mechanism of VIAAT remains largely unknown. Here, we show that proteoliposomes containing purified VIAAT actively took up GABA upon formation of membrane potential (⌬) (positive inside) but not ⌬pH. VIAAT-mediated GABA uptake had an absolute requirement for Cl ؊ and actually accompa-

Unique pH dynamics in GABAergic synaptic vesicles illuminates the mechanism and kinetics of GABA loading

“…In addition, acidification of GABAergic SVs must precede GABA uptake (i.e., protonation of the luminal side of VGAT is required for its activation). Such observations are in good agreement with earlier biochemical studies of GABA uptake into isolated SVs (9) that suggested a pivotal contribution of ΔpH on GABA transport and argue against an alternative model of ΔΨ-driven GABA/2Cl -cotransport (13). The latter mode of coupling seems unlikely in intact SVs, because Cl -entry coupled to GABA transport would promote acidification by dissipating ΔΨ (38).…”

“…We hypothesized that SV refilling with GABA, which has been proposed to occur through a GABA/H + exchange (9) (ref. 13 shows an alternative model), may underlie the observed SV alkalization. To address this hypothesis, we monitored the pH of SVs from VGAT-deficient neurons.…”

“…-cotransporter without the need for ΔpH (13). Clarification of the enigmatic GABA transport mechanism was recently provided by Farsi et al (14), who imaged the pH-sensitive superecliptic pHluorin in isolated single SVs and found evidence that VGAT functions through a GABA/H + antiport mechanism.…”

Type 1 Sodium-dependent Phosphate Transporter (SLC17A1 Protein) Is a Cl−-dependent Urate Exporter

“…Under similar assay conditions, VGAT that is known to cotransport 2 Cl Ϫ and GABA (17), took up radiolabeled Cl Ϫ in the presence of GABA (Fig. 4B) (17). Furthermore, only the background level of Cl Ϫ uptake was observed by NPT1-containing proteoliposomes entrapped with PAH at 1 mM (data not shown).…”

“…2B). We then tested whether Cl Ϫ protected DIDS-mediated inactivation because Cl Ϫ competes with DIDS in VGLUT, suggesting the same binding site(s) for Cl Ϫ and DIDS (12,16,17). We found that DIDS strongly inhibited ⌬-dependent PAH uptake with an ID 50 of 5 M (Fig.…”