Allosteric Inhibition of a Vesicular Glutamate Transporter by an Isoform-Specific Antibody.

The role of glutamate in excitatory neurotransmission depends on its transport into synaptic vesicles by the vesicular glutamate transporters (VGLUTs). The three VGLUT isoforms exhibit a complementary distribution in the nervous system, and the knockout of each produces severe, pleiotropic neurological effects. However, the available pharmacology lacks sensitivity and specificity, limiting the analysis of both transport mechanism and physiological role. To develop new molecular probes for the VGLUTs, we raised six mouse monoclonal antibodies to VGLUT2. All six bind to a structured region of VGLUT2, five to the luminal face, and one to the cytosolic. Two are specific to VGLUT2, whereas the other four bind to both VGLUT1 and 2; none detect VGLUT3. Antibody 8E11 recognizes an epitope spanning the three extracellular loops in the C-domain that explains the recognition of both VGLUT1 and 2 but not VGLUT3. 8E11 also inhibits both glutamate transport and the VGLUT-associated chloride conductance. Since the antibody binds outside the substrate recognition site, it acts allosterically to inhibit function, presumably by restricting conformational changes. The isoform specificity also shows that allosteric inhibition provides a mechanism to distinguish between closely related transporters.

Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of Transport.

The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.

Vesicular glutamate transporters use flexible anion and cation binding sites for efficient accumulation of neurotransmitter.

Vesicular glutamate transporters (VGLUTs) accumulate the neurotransmitter glutamate in synaptic vesicles. Transport depends on a V-ATPase-dependent electrochemical proton gradient (ΔµH+) and requires chloride ions, but how chloride acts and how ionic and charge balance is maintained during transport is controversial. Using a reconstitution approach, we used an exogenous proton pump to drive VGLUT-mediated transport either in liposomes containing purified VGLUT1 or in synaptic vesicles fused with proton-pump-containing liposomes. Our data show that chloride stimulation can be induced at both sides of the membrane. Moreover, chloride competes with glutamate at high concentrations. In addition, VGLUT1 possesses a cation binding site capable of binding H+ or K+ ions, allowing for proton antiport or K+ / H+ exchange. We conclude that VGLUTs contain two anion binding sites and one cation binding site, allowing the transporter to adjust to the changing ionic conditions during vesicle filling without being dependent on other transporters or channels.

Unveiling the secret lives of glutamate transporters: VGLUTs engage in multiple transport modes.

Accumulation of glutamate in synaptic vesicles is mediated by vesicular glutamate transporters called VGLUTs. In the current issue of Neuron, Preobraschenski et al. (2014) show that the VGLUTs, in addition to transporting glutamate, also provide the conductances necessary to maintain the appropriate voltage and pH inside these vesicles.

Distribution of gephyrin-immunoreactivity in the trigeminal motor nucleus: an immunohistochemical study in rats.

It has been established that a postsynaptic scaffolding protein, gephyrin, is essential for anchoring two main groups of inhibitory receptors, GABA(A) receptors (GABA(A) Rs) and glycine receptors (GlyRs), to the postsynaptic sites of neurons. The present study was primarily attempted to examine if expression patterns of gephyrin might be different between jaw-closing (JC) and jaw-opening (JO) motoneurons. The JC- and JO-motoneurons in the rat trigeminal motor nucleus (Vm) were located in the dorsolateral (Vm.dl) and ventromedial (Vm.vm) divisions, respectively (Mizuno et al.,1975). Thus, immunoreactivity (IR) for gephyrin was investigated in the Vm: immunofluorescence histochemistry for gephyrin was combined with retrograde tract-tracing of fluorogold (FG), which was injected into nerves innervating JC-muscles or nerves innervating JO-muscles; neuronal cells were counterstained with propidium iodide (PI). The Vm.dl was discriminated from the Vm.vm by the presence of vesicular glutamate transporter 1 (VGLUT1)-immunopositive axon terminals, which were distributed in the Vm.dl but not in the Vm.vm (Pang et al., J Comp Neurol 2009;512:595-612). Gephyrin-IR showed a punctate pattern of fluorescence, and motoneuronal profiles were coated with small clusters of gephyrin-immunopositive puncta throughout the Vm. The distribution density of such clusters was apparently higher in the Vm.dl than in the Vm.vm; this was confirmed quantitatively by a method similar to that described by Lorenzo et al. (Eur J Neurosci 2006;23:3161-3170). On the basis of the present results, possible correlation between the distribution density of gephyrin clusters in the submembrane region of Vm motoneurons and that of axon terminals making inhibitory synapses on Vm motoneurons was discussed.

Docking and homology modeling explain inhibition of the human vesicular glutamate transporters.

As membrane transporter proteins, VGLUT1-3 mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells. This function is crucial for exocytosis and the role of glutamate as the major excitatory neurotransmitter in the central nervous system. The three transporters, sharing 76% amino acid sequence identity in humans, are highly homologous but differ in regional expression in the brain. Although little is known regarding their three-dimensional structures, hydropathy analysis on these proteins predicts 12 transmembrane segments connected by loops, a topology similar to other members in the major facilitator superfamily, where VGLUT1-3 have been phylogenetically classified. In this work, we present a three-dimensional model for the human VGLUT1 protein based on its distant bacterial homolog in the same superfamily, the glycerol-3-phosphate transporter from Escherichia coli. This structural model, stable during molecular dynamics simulations in phospholipid bilayers solvated by water, reveals amino acid residues that face its pore and are likely to affect substrate translocation. Docking of VGLUT1 substrates to this pore localizes two different binding sites, to which inhibitors also bind with an overall trend in binding affinity that is in agreement with previously published experimental data.

Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling.

Synaptic vesicles have been proposed to form through two mechanisms: one directly from the plasma membrane involving clathrin-dependent endocytosis and the adaptor protein AP2, and the other from an endosomal intermediate mediated by the adaptor AP3. However, the relative role of these two mechanisms in synaptic vesicle recycling has remained unclear. We now find that vesicular glutamate transporter VGLUT1 interacts directly with endophilin, a component of the clathrin-dependent endocytic machinery. In the absence of its interaction with endophilin, VGLUT1 recycles more slowly during prolonged, high-frequency stimulation. Inhibition of the AP3 pathway with brefeldin A rescues the rate of recycling, suggesting a competition between AP2 and -3 pathways, with endophilin recruiting VGLUT1 toward the faster AP2 pathway. After stimulation, however, inhibition of the AP3 pathway prevents the full recovery of VGLUT1 by endocytosis, implicating the AP3 pathway specifically in compensatory endocytosis.

Interaction between the vesicular glutamate transporter type 1 and endophilin A1, a protein essential for endocytosis.

In the nerve terminal, neurotransmitter is actively packaged into synaptic vesicles before its release by Ca2+-dependent exocytosis. The three vesicular glutamate transporters (VGLUT1, -2 and -3) are highly conserved proteins that display similar bioenergetic and pharmacological properties but are expressed in different brain areas. We used the divergent C-terminus of VGLUT1 as a bait in a yeast two-hybrid screen to identify and map the interaction between a proline-rich domain of VGLUT1 and the Src homology domain 3 (SH3) domain of endophilin. We further confirmed this interaction by using different glutathione-S-transferase-endophilin fusion proteins to pull down VGLUT1 from rat brain extracts. The expression profiles of the two genes and proteins were compared on rat brain sections, showing that endophilin is most highly expressed in regions and cells expressing VGLUT1. Double immunofluorescence in the rat cerebellum shows that most VGLUT1-positive terminals co-express endophilin, whereas VGLUT2-expressing terminals are often devoid of endophilin. However, neither VGLUT1 transport activity, endophilin enzymatic activity nor VGLUT1 synaptic targeting were altered by this interaction. Overall, the discovery of endophilin as a partner for VGLUT1 in nerve terminals strongly suggests the existence of functional differences between VGLUT1 and -2 terminals in their abilities to replenish vesicle pools.