Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In this study, we show that VGAT recognizes β-alanine as a substrate. Proteoliposomes containing purified VGAT transport β-alanine using Δψ but not ΔpH as a driving force. The Δψ-driven β-alanine uptake requires Cl−. VGAT also facilitates Cl− uptake in the presence of β-alanine. A previously described VGAT mutant (Glu213Ala) that disrupts GABA and glycine transport similarly abrogates β-alanine uptake. These findings indicated that VGAT transports β-alanine through a mechanism similar to those for GABA and glycine, and functions as a vesicular β-alanine transporter.
Vesicular GABA transporter (VGAT) is expressed in GABAergic and glycinergic neurons, and is responsible for vesicular storage and subsequent exocytosis of these inhibitory amino acids. In the present study, we showed that proteoliposomes containing purified VGAT transport β-alanine using Δψ as a driving force. VGAT also facilitates Cl− uptake. Our findings indicated that VGAT functions as a vesicular β-alanine transporter.
Before exocytosis classical neurotransmitters are stored in secretory vesicles through active transport at the expense of an electrochemical gradient of protons across the membrane established by vacuolar proton ATPase (Nelson and Harvey 1999). Six classes of vesicular neurotransmitter transporters are responsible for the vesicular storage of classical neurotransmitters (Omote et al. 2011; Omote and Moriyama 2013). Vesicular GABA transporter (VGAT) or vesicular inhibitory amino acid transporter (VIAAT) plays an essential role in the vesicular storage of GABA and glycine in GABAergic and glycinergic neurons, respectively, and is involved in inhibitory neurotransmission (McIntire et al. 1997; Chaudhry et al. 1998; Bedet et al. 2000; Gasnier 2004). VGAT knockout mice exhibited a lack of vesicular storage of GABA and glycine and impaired inhibitory neurotransmission (Wojcik et al. 2006). A recent study involving purified VGAT demonstrated that VGAT functions as a GABA (glycine) and 2 Cl− co-transporter and that the membrane potential (Δψ) drives the uptake (Juge et al. 2009). Furthermore, a glutamate residue at the position 213 is essential for GABA and glycine transport (Juge et al. 2009).
β-Alanine is one of the naturally occurring β-amino acids. β-Alanine is released from neurons through a Ca2+-dependent processes (Sandberg and Jacobson 1981). Once released, β-alanine may bind to the GABAA receptor and hyperpolarize the target cells (Barker et al. 1982). β-Alanine also binds to the GABAC receptor, and elicits an inhibitory response similar to GABA-evoked ones (Mori et al. 2002). Furthermore, β-alanine binds to the glycine binding site on the NMDA receptor and modulates its activity (Rajendra et al. 1997). β-Alanine also binds to the strychnine-sensitive glycine receptor and activates it (Betz 1991). Extracellular β-alanine in turn is taken up by plasma membrane GABA transporters, GAT2, GAT3 or GAT4 to terminate inhibitory neurotransmission (Liu et al. 1993). Although the combined evidence supports the idea that β-alanine acts as an inhibitory neurotransmitter, very important mechanistic evidence is still missing: the vesicular transporter(s) that is responsible for the vesicular storage of β-alanine remains unclear.
In this work, we showed direct evidence that β-alanine is a transport substrate of VGAT.
Purification of wild-type and mutant VGATs
Wild-type VGAT and mutant VGATs harboring Glu213Ala or Lys351Ala were expressed in High Five cells, and purified as described previously (Juge et al. 2009). The Lys351Ala mutation was introduced using the primer 5′-ACATCGCCGCCTGCGTGCTCGCGGGTC-3′. The purified VGATs were stored at −80°C, at which it was stable without loss of activity for at least a few months.
Purified VGAT and bacterial F-ATPase were co-reconstituted into liposomes by the freeze-thaw method (Juge et al. 2009). In brief, 10 μg of eluate protein (purified VGAT fraction) was mixed with or without 90 μg of F-ATPase and asolectin liposomes (0.5 mg), frozen at −80°C and then left at this temperature for at least 5 min. Then the mixture was thawed quickly and diluted 60-fold with the buffer containing 20 mM 3-morpholinopropane sulfonic acid (MOPS)-Tris, pH 7.0, 0.5 mM dithiothreitol, 0.15 M sodium acetate and 5 mM magnesium acetate. After sedimentation, the VGAT-containing proteoliposomes were suspended in 0.4 mL of 20 mM MOPS-Tris, pH 7.0, containing 0.15 M sodium acetate and 5 mM magnesium acetate. Asolectin liposomes were prepared as follows. Soybean lecithin (20 mg; Sigma type IIS, Okayama, Japan) was suspended in 2 mL of 20 mM MOPS-NaOH, pH 7.0 containing 0.5 mM dithiothreitol. The mixture was sonicated in a bath-type sonicator until clear, divided into small aliquots, and stored at −80°C until use.
Proteoliposomes (5 μg of total protein/assay) were suspended in 20 mM MOPS-Tris, pH 7.5, 5 mM magnesium acetate, 4 mM KCl and 0.1 M potassium acetate, and then incubated for 3 min at 27°C. ATP was added to give a final concentration of 2 mM and the mixture was incubated for a further 3 min. The assay was initiated by the addition of 100 μM [2,3-3H] GABA (0.5 MBq/μmol), 2 mM [3H] β-alanine (0.5 MBq/μmol), or 2 mM [1,2-3H] taurine (0.5 MBq/μmol), and 130 μL aliquots were taken at the times indicated and centrifuged through a Sephadex G-50 (fine) spin column at 760 g for 2 min. For valinomycin (Val)-induced transport assays, proteoliposomes containing VGAT (0.5 μg of total protein/assay) were suspended in 0.5 mL of 20 mM MOPS-Tris, pH 7.0, 5 mM magnesium acetate, 10 mM KCl and 0.15 M potassium acetate, and then incubated for 3 min at 27°C. Valinomycin was added to a final concentration of 2 μM, and the mixture was incubated for a further 3 min. A radiolabeled β-alanine was then added to a final concentration of 2 mM [3H] β-alanine (0.5 MBq/μmol), to initiate assays. Radiolabeled Cl− uptake assay was carried out as described previously (Juge et al. 2009). Radiolabeled 36Cl− (740 MBq/g) was used as a substrate at a final concentration of 5 or 10 mM.
Materials and miscellaneous procedures
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and western blotting were performed as described (Juge et al. 2009). Bacterial F-ATPase was expressed in DK8/pBWU13 Escherichia coli cells and purified by glycerol density gradient centrifugation as described (Moriyama et al. 1991). Protein concentration was determined using bovine serum albumin as a standard (Schaffner and Weissmann 1973).
Results and Discussion
VGAT transports β-alanine
We measured β-alanine uptake by proteoliposomes containing purified VGAT and F-ATPase. Upon the addition of ATP, the proteoliposomes established an electrochemical gradient of protons across the membranes to facilitate the uptake of β-alanine. It was found that the proteoliposomes took up β-alanine in a time- and ATP-dependent manner (Fig. 1a). Both VGAT and F-ATPase are necessary for the complete uptake of β-alanine, because the absence of VGAT or F-ATPase, or both led to only a background level of β-alanine uptake. The uptake of β-alanine exhibited concentration dependence of β-alanine with Km and Vmax of 3.5 mM and 38 nmol/min/mg, respectively (Fig. 1b). In contrast, no ATP-dependent uptake of taurine, a sulfonyl form of β-alanine, by the proteoliposomes containing VGAT and F-ATPase was observed. Consistently, VGAT-mediated GABA uptake was inhibited by β-alanine but not by taurine (Fig. 1c). These results indicated that VGAT transports β-alanine.
Characteristics of VGAT-mediated β-alanine uptake
We further characterized the VGAT-mediated β-alanine transport. The ATP-dependent β-alanine uptake was inhibited by sodium azide, an inhibitor of F-ATPase, but not by bafilomycin A1, an inhibitor of V-ATPase (Nelson and Harvey 1999), confirming that F-ATPase supplies the driving force for the uptake. ATP-dependent β-alanine uptake was sensitive to carbonyl cyanide 3-chlorophenylhydrazone (CCCP), indicating that ΔμH+ drove the uptake. Valinomycin reduced the β-alanine uptake to 43% of the control level, while nigericin in the presence of K+ had a limited effect. The combination of valinomycin and nigericin abolished the activity. Furthermore, ammonium sulfate, a dissipater of ΔpH but not of Δψ, slightly decreased the β-alanine uptake. These results suggested that VGAT preferentially used Δψ but not ΔpH as the driving force for the uptake of β-alanine as in VGAT-mediated GABA or glycine transport.
VGAT possesses nine or ten transmembrane spanning helices, in which several charged amino acid residues are located (McIntire et al. 1997). Our previous study indicated that VGAT with the Glu213 to Ala substitution exhibited an almost complete lack of transport of GABA and glycine (Juge et al. 2009). As shown in Fig. 2b, the Glu213Ala mutant had lost the ability of β-alanine uptake, while the Lys351Ala mutant retained around 80% of the transport activity. Consistently, the Lys351Ala VGAT exhibited similar kinetics as wild type with Km and Vmax values of 3.1 mM and 30 nmol/min/mg for β-alanine and 1.0 mM and 50 nmol/min/mg for GABA, respectively.
One of the peculiar properties of VGAT-mediated GABA and glycine uptake is its mechanistic coupling to Cl−: VGAT requires Cl− for its glycine and GABA uptake, and co-transports one GABA (glycine) with 2 Cl−. Thus, we investigated whether or not VGAT co-transports β-alanine and Cl−. To measure the co-transport of β-alanine and Cl−, we prepared proteoliposomes containing only purified VGAT as a protein source in the absence of K+. The proteoliposomes were suspended in the buffer containing K+ and Δψ was formed across the membrane upon the addition of valinomycin around 90 mV (positive inside). Under the conditions, we can measure the uptake of β-alanine and Cl− under the steady driving force (Juge et al. 2009). As shown in Fig. 2c, valinomycin-induced formation of Δψ drove the β-alanine uptake in the presence of Cl−, confirming the driving force and Cl−-dependent β-alanine uptake by VGAT. The uptake of radiolabeled Cl− was also driven by Δψ in the presence of β-alanine. In the absence of β-alanine, the uptake of Cl− decreased to the background level (Fig. 2d).
VGAT is a vesicular β-alanine transporter
In spite of the well accepted receptor activity and sequestration of the extracellular space, little is known about the mechanism of vesicular storage of β-alanine. Until now, only a few papers described the β-alanine uptake into synaptic vesicles (Christensen et al. 1991; Roseth and Fonnum 1995). Based on the cis-inhibition of ATP-dependent GABA uptake with cold β-alanine, Fonnum and his colleague suggested that β-alanine is a transport substrate for ATP-dependent GABA transport in synaptic vesicles (Roseth and Fonnum 1995). No further evidence for characterization of vesicular β-alanine transport is available. In this study, we demonstrated that purified VGAT transports β-alanine with essentially the same properties to those of GABA and glycine transport as to driving force, mode of coupling to Cl− and effect of mutation. Taking the Km and Vmax values into consideration, the present results strongly suggested that VGAT functions as vesicular β-alanine transporter and is responsible for vesicular storage of β-alanine under physiological condition.
VGAT-mediated β-alanine transport is driven by Δψ and co-transported with Cl−. However, the result does not rule out the possible participation of ΔpH (or internal acidic pH) in β-alanine transport, since the selective dissipation of ΔpH by ammonium ions or nigericin in the presence of K+ gave slightly inhibitory effect, as in the case with VGAT-mediated uptakes of GABA and glycine (Juge et al. 2009; Omote and Moriyama 2013). More detailed study will be necessary to determine the role of ΔpH (or internal acidic pH) in VGAT-mediated transport.
It should be emphasized that the present study identified at least in part the molecular machinery for vesicular β-alanine storage, and thus supports the occurrence of chemical transmission by β-alanine in vivo. It also suggests that VGAT-expressing neurons may secrete not only GABA and glycine but also β-alanine and use them as inhibitory neurotransmitters. Future studies, in particular, identification of secretory organelles containing both β-alanine and VGAT and detection of VGAT-mediated vesicular storage and secretion of β-alanine will be necessary to conclude occurrence of β-alanine-mediated neurotransmission.
This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan for Young Scientists (B) to NJ; Grants-in Aid for Scientific Research (B) to HO; and Grants-in-Aid for Scientific Research (A), the Salt Science Foundation (no.1139), and Japan Science and Technology Agency for Japan-Israel Scientific Research Cooperation to YM. The authors have no conflict of interest to declare.