Abstract
The vesicular inhibitory amino acid transporter (VIAAT) is a synaptic vesicle protein responsible for the vesicular storage of gamma-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 (Deltapsi) (positive inside) but not DeltapH. VIAAT-mediated GABA uptake had an absolute requirement for Cl(-) and actually accompanied Cl(-) movement. Kinetic analysis indicated that one GABA molecule and two Cl(-) equivalents were transported during one transport cycle. VIAAT in which Glu(213) was specifically mutated to alanine completely lost the ability to take up both GABA and Cl(-). Essentially the same results were obtained with glycine, another substrate of VIAAT. These results demonstrated that VIAAT is a vesicular Cl(-) transporter that co-transports Cl(-) with GABA or glycine in a Deltapsi dependent manner. It is concluded that Cl(-) plays an essential role in vesicular storage of GABA and glycine.
Highlights
Background level ofGABA transport activity was observed in liposomes containing no vesicular inhibitory amino acid transporter (VIAAT).Consistent with proteoliposomes containing both VIAAT and F-ATPase, GABA transport was not observed when ClϪ was omitted from the assay buffer of VIAAT-containing proteoliposomes even though the maximum level of ⌬ was maintained (Fig. 2B)
Western blot analysis revealed that the upper two polypeptides corresponded to VIAAT (Fig. 1A, lane 8), which is consistent with previous observations [14, 21]
We found that ClϪ was absolutely required for VIAAT activity: essentially no transport activity was obE213A mutant as one of the negative controls
Summary
Background level ofGABA transport activity was observed in liposomes containing no VIAAT.Consistent with proteoliposomes containing both VIAAT and F-ATPase, GABA transport was not observed when ClϪ was omitted from the assay buffer of VIAAT-containing proteoliposomes even though the maximum level of ⌬ was maintained (Fig. 2B). For ATP-driven transport, proteoliposomes containing both VIAAT and F-ATPase (5 g of total protein) were suspended in 20 mM MOPS-Tris (pH 7.5), 5 mM magnesium acetate, 4 mM KCl, and 0.1 M potassium acetate and incubated for 3 min at 27 °C. For Val-induced transport assays, proteoliposomes containing VIAAT (0.5 g of protein/assay) were suspended in 500 l of 20 mM MOPS-Tris (pH 7.0), 5 mM magnesium acetate, 10 mM KCl, and 0.15 M potassium acetate and incubated for 3 min at 27 °C.
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