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.

Ion transport and regulation in a synaptic vesicle glutamate transporter.

Synaptic vesicles accumulate neurotransmitters, enabling the quantal release by exocytosis that underlies synaptic transmission. Specific neurotransmitter transporters are responsible for this activity and therefore are essential for brain function. The vesicular glutamate transporters (VGLUTs) concentrate the principal excitatory neurotransmitter glutamate into synaptic vesicles, driven by membrane potential. However, the mechanism by which they do so remains poorly understood owing to a lack of structural information. We report the cryo-electron microscopy structure of rat VGLUT2 at 3.8-angstrom resolution and propose structure-based mechanisms for substrate recognition and allosteric activation by low pH and chloride. A potential permeation pathway for chloride intersects with the glutamate binding site. These results demonstrate how the activity of VGLUTs can be coordinated with large shifts in proton and chloride concentrations during the synaptic vesicle cycle to ensure normal synaptic transmission.

Liraglutide Modulates Appetite and Body Weight Through Glucagon-Like Peptide 1 Receptor-Expressing Glutamatergic Neurons.

Glucagon-like peptide 1 receptor (GLP-1R) agonists are U.S. Food and Drug Administration-approved weight loss drugs. Despite their widespread use, the sites of action through which GLP-1R agonists (GLP1RAs) affect appetite and body weight are still not fully understood. We determined whether GLP-1Rs in either GABAergic or glutamatergic neurons are necessary for the short- and long-term effects of the GLP1RA liraglutide on food intake, visceral illness, body weight, and neural network activation. We found that mice lacking GLP-1Rs in vGAT-expressing GABAergic neurons responded identically to controls in all parameters measured, whereas deletion of GLP-1Rs in vGlut2-expressing glutamatergic neurons eliminated liraglutide-induced weight loss and visceral illness and severely attenuated its effects on feeding. Concomitantly, deletion of GLP-1Rs from glutamatergic neurons completely abolished the neural network activation observed after liraglutide administration. We conclude that liraglutide activates a dispersed but discrete neural network to mediate its physiological effects and that these effects require GLP-1R expression on glutamatergic but not GABAergic neurons.

Optogenetic inactivation of the subthalamic nucleus improves forelimb akinesia in a rat model of Parkinson disease.

BACKGROUND: The inhibition of neuronal activity by electrical deep brain stimulation is one of the mechanisms explaining the therapeutic effects in patients with Parkinson disease (PD) but cannot specifically activate or inactivate different types of neurons. Recently, a new technology based on optogenetics has been developed to modulate the activity of specific neurons. However, the therapeutic effects of optical inactivation in the subthalamic nucleus (STN) have not been fully investigated. OBJECTIVE: To perform various behavioral tests to evaluate changes in motor functions in a PD rat model after optogene expression and, unlike previous studies, to assess the therapeutic effects of direct optogenetic inactivation in the STN. METHODS: 6-Hydroxydopamine-induced hemiparkinsonian rats received injections of hSynapsin1-NpHR-YFP adeno-associated virus or an equivalent volume of phosphate-buffered saline. Three weeks after injection of adeno-associated virus or phosphate-buffered saline, the optic fiber was implanted into the ipsilateral STN. A stepping test, a cylinder test, and an apomorphine-induced rotation test were performed in 3 sequential steps: during light-off state, during light stimulation, and again during light-off state. RESULTS: Stepping tests revealed that optical inhibition of the STN significantly improved 6-hydroxydopamine-induced forelimb akinesia. PD motor signs, as assessed by cylinder and apomorphine tests, were not affected by optical inhibition. Immunofluorescence revealed that halorhodopsin was highly expressed and colocalized with vesicular glutamate transporter 2 in the STN. CONCLUSION: Optogenetic inhibition in the STN may be effective in improving contralateral forelimb akinesia but not in changing forelimb preference or reducing dopaminergic receptor supersensitivity. These findings are useful as a basis for future studies on optogenetics in PD.

Metabolic control of vesicular glutamate transport and release.

Fasting has been used to control epilepsy since antiquity, but the mechanism of coupling between metabolic state and excitatory neurotransmission remains unknown. Previous work has shown that the vesicular glutamate transporters (VGLUTs) required for exocytotic release of glutamate undergo an unusual form of regulation by Cl(-). Using functional reconstitution of the purified VGLUTs into proteoliposomes, we now show that Cl(-) acts as an allosteric activator, and the ketone bodies that increase with fasting inhibit glutamate release by competing with Cl(-) at the site of allosteric regulation. Consistent with these observations, acetoacetate reduced quantal size at hippocampal synapses and suppresses glutamate release and seizures evoked with 4-aminopyridine in the brain. The results indicate an unsuspected link between metabolic state and excitatory neurotransmission through anion-dependent regulation of VGLUT activity.

Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins.

The rate at which X-ray structures of membrane proteins are solved is on a par with that of soluble proteins in the late 1970s. There are still many obstacles facing the membrane protein structural community. Recently, there have been several technical achievements in the field that have started to dramatically accelerate structural studies. Here, we summarize these so-called 'tricks-of-the-trade' and include case studies of several mammalian transporters.

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.

Vesicular glutamate transporter contains two independent transport machineries.

Vesicular glutamate transporters (VGLUTs) are responsible for the vesicular storage of l-glutamate and play an essential role in glutamatergic signal transmission in the central nervous system. The molecular mechanism of the transport remains unknown. Here, we established a novel in vitro assay procedure, which includes purification of wild and mutant VGLUT2 and their reconstitution with purified bacterial F(o)F(1)-ATPase (F-ATPase) into liposomes. Upon the addition of ATP, the proteoliposomes facilitated l-glutamate uptake in a membrane potential (DeltaPsi)-dependent fashion. The ATP-dependent l-glutamate uptake exhibited an absolute requirement for approximately 4 mm Cl(-), was sensitive to Evans blue, but was insensitive to d,l-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg(184), His(128), and Glu(191) showed a dramatic loss in l-glutamate transport activity, whereas Na(+)-dependent inorganic phosphate (P(i)) uptake remained comparable to that of the wild type. Furthermore, P(i) transport did not require Cl(-) and was not inhibited by Evans blue. Thus, VGLUT2 appears to possess two intrinsic transport machineries that are independent of each other: a DeltaPsi-dependent l-glutamate uptake and a Na(+)-dependent P(i) uptake.