Language：English   Publishing type：Research paper (scientific journal)  

OBJECTIVE: The ketogenic diet is clinically used to treat drug-resistant epilepsy. The diet treatment markedly increases ketone bodies (acetoacetate and β-hydroxybutyrate), which work as energy metabolites in the brain. Here, we investigated effects of acetoacetate on voltage-dependent Ca2+ channels (VDCCs) in pyramidal cells of the hippocampus. We further explored an acetoacetate analog that inhibited VDCCs in pyramidal cells, reduced excitatory postsynaptic currents (EPSCs), and suppressed seizures in vivo. METHODS: The effects of acetoacetate and its analogs on VDCCs and EPSCs were evaluated using patch-clamp recordings from CA1 pyramidal cells of mouse hippocampal slices. The in vivo effects of these reagents were also evaluated using a chronic seizure model induced by intrahippocampal injection of kainate. RESULTS: Acetoacetate inhibited VDCCs in pyramidal cells of hippocampal slices, and reduced EPSCs in slices exhibiting epileptiform activity. More potent EPSC inhibitors were then explored by modifying the chemical structure of acetoacetate, and 2-phenylbutyrate was identified as an acetoacetate analog that inhibited VDCCs and EPSCs more potently. Although acetoacetate is known to inhibit vesicular glutamate transporters (VGLUTs), 2-phenylbutyrate did not inhibit VGLUTs, showing that 2-phenylbutyrate is an acetoacetate analog that preferably inhibits VDCCs. In addition, 2-phenylbutyrate markedly reduced EPSCs in slices exhibiting epileptiform activity, and suppressed hippocampal seizures in vivo in a mouse model of epilepsy. The in vivo antiseizure effects of 2-phenylbutyrate were more potent than those of acetoacetate. Finally, intraperitoneal 2-phenylbutyrate was delivered to the brain, and its brain concentration reached the level enough to reduce EPSCs. SIGNIFICANCE: These results demonstrate that 2-phenylbutyrate is an acetoacetate analog that inhibits VDCCs and EPSCs in pyramidal cells, suppresses hippocampal seizures in vivo, and has brain penetration ability. Thus 2-phenylbutyrate provides a useful chemical structure as a lead compound to develop new antiseizure drugs originating from ketone bodies.

DOI： 10.1111/epi.13718

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Vesicular neurotransmitter transporters are responsible for the accumulation of neurotransmitters in secretory vesicles and play essential roles in chemical transmission. The SLC17 family contributes to sequestration of anionic neurotransmitters such as glutamate, aspartate, and nucleotides. Identification and subsequent cellular and molecular biological studies of SLC17 transporters unveiled the principles underlying the actions of these transporters. Recent progress in reconstitution methods in combination with postgenomic approaches has advanced studies on neurotransmitter transporters. This review summarizes the molecular properties of SLC17-type transporters and recent findings regarding the novel SLC18 transporter.

DOI： 10.1146/annurev-pharmtox-010814-124816

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Membrane potential (Δψ)-driven and Cl(-)-dependent organic anion transport is a primary function of the solute carrier family 17 (SLC17) transporter family. Although the transport substrates and physiological relevance of the major members are well understood, SLC17A2 protein known to be Na(+)-phosphate cotransporter 3 (NPT3) is far less well characterized. In the present study, we investigated the transport properties and expression patterns of mouse SLC17A2 protein (mNPT3). Proteoliposomes containing the purified mNPT3 protein took up radiolabeled p-aminohippuric acid (PAH) in a Δψ- and Cl(-)-dependent manner. The mNPT3-mediated PAH uptake was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDs) and Evans blue, common inhibitors of SLC17 family members. The PAH uptake was also inhibited by various anionic compounds, such as hydrophilic nonsteroidal anti-inflammatory drugs (NSAIDs) and urate. Consistent with these observations, the proteoliposome took up radiolabeled urate in a Δψ- and Cl(-)-dependent manner. Immunohistochemistry with specific antibodies against mNPT3 combined with RT-PCR revealed that mNPT3 is present in various tissues, including the hepatic bile duct, luminal membranes of the renal urinary tubules, maternal side of syncytiotrophoblast in the placenta, apical membrane of follicle cells in the thyroid, bronchiole epithelial cells in the lungs, and astrocytes around blood vessels in the cerebrum. These results suggested that mNPT3 is a polyspecific organic anion transporter that is involved in circulation of urate throughout the body.

DOI： 10.1152/ajpcell.00048.2015

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Extrusion of chloroquine (CQ) from digestive vacuoles through the Plasmodium falciparum CQ resistance transporter (PfCRT) is essential to establish CQ resistance of the malaria parasite. However, the physiological relevance of PfCRT and how CQ-resistant PfCRT gains the ability to transport CQ remain unknown. We prepared proteoliposomes containing purified CQ-sensitive and CQ-resistant PfCRTs and measured their transport activities. All PfCRTs tested actively took up tetraethylammonium, verapamil, CQ, basic amino acids, polypeptides, and polyamines at the expense of an electrochemical proton gradient. CQ-resistant PfCRT exhibited decreased affinity for CQ, resulting in increased CQ uptake. Furthermore, CQ competitively inhibited amino acid transport. Thus, PfCRT is a H(+)-coupled polyspecific nutrient and drug exporter.

DOI： 10.1073/pnas.1417102112

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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.

DOI： 10.1111/jnc.12393

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In spite of its recent achievements, the technique of single particle electron cryomicroscopy (cryoEM) has not been widely used to study proteins smaller than 100 kDa, although it is a highly desirable application of this technique. One fundamental limitation is that images of small proteins embedded in vitreous ice do not contain adequate features for accurate image alignment. We describe a general strategy to overcome this limitation by selecting a fragment antigen binding (Fab) to form a stable and rigid complex with a target protein, thus providing a defined feature for accurate image alignment. Using this approach, we determined a three-dimensional structure of an ∼65 kDa protein by single particle cryoEM. Because Fabs can be readily generated against a wide range of proteins by phage display, this approach is generally applicable to study many small proteins by single particle cryoEM.

DOI： 10.1016/j.str.2012.02.017

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Glutamate plays essential roles in chemical transmission as a major excitatory neurotransmitter. The accumulation of glutamate in secretory vesicles is mediated by vesicular glutamate transporters (VGLUTs) that together with the driving electrochemical gradient of proteins influence the subsequent quantum release of glutamate and the function of higher-order neurons. The vesicular content of glutamate is well correlated with membrane potential (Δψ), which suggests that Δψ determines the vesicular glutamate concentration. The transport of glutamate into secretory vesicles is highly dependent on Cl(-). This anion stimulates glutamate transport but is inhibitory at higher concentrations. Accumulating evidence indicates that Cl(-) regulates glutamate transport through control of VGLUT activity and the H(+) electrochemical gradient. Recently, a comprehensive study demonstrated that Cl(-) regulation of VGLUT is competitively inhibited by metabolic intermediates such as ketone bodies. It also showed that ketone bodies are effective in controlling epilepsy. These results suggest a correlation between metabolic state and higher-order brain function. We propose a novel function for Cl(-) as a fundamental regulator for signal transmission.

DOI： 10.1021/bi200567k

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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.

DOI： 10.1016/j.neuron.2010.09.002

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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.

DOI： 10.1074/jbc.M109.062414

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Authorship：Lead author,　Last author,　Corresponding author   Publishing type：Part of collection (book)  

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ATP is a major chemical transmitter in purinergic signal transmission. Before secretion, ATP is stored in secretory vesicles found in purinergic cells. Although the presence of active transport mechanisms for ATP has been postulated for a long time, the proteins responsible for its vesicular accumulation remains unknown. The transporter encoded by the human and mouse SLC17A9 gene, a novel member of an anion transporter family, was predominantly expressed in the brain and adrenal gland. The mouse and bovine counterparts were associated with adrenal chromaffin granules. Proteoliposomes containing purified transporter actively took up ATP, ADP, and GTP by using membrane potential as the driving force. The uptake properties of the reconstituted transporter were similar to that of the ATP uptake by synaptic vesicles and chromaffin granules. Suppression of endogenous SLC17A9 expression in PC12 cells decreased exocytosis of ATP. These findings strongly suggest that SLC17A9 protein is a vesicular nucleotide transporter and should lead to the elucidation of the molecular mechanism of ATP secretion in purinergic signal transmission.

DOI： 10.1073/pnas.0800141105

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Last author,　Corresponding author  

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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.

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Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L-glutamate. Osteoclasts secrete L-glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+-dependent manner. KCl- and ATP-dependent secretion of L-glutamate was absent in osteoclasts prepared from VGLUT1-/- knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L-glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1-/- mice develop osteoporosis. Thus, in bone-resorbing osteoclasts, L-glutamate and bone degradation products are secreted through transcytosis and the released L-glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.

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Vesicular glutamate transporter (VGLUT) is responsible for the vesicular storage of l-glutamate, and plays an essential role in glutamate-mediated intercellular signal transmission in the CNS and in some neuroendocrine cells. Intestinal L cells are the glucose-responsive neuroendocrine cells responsible for the secretion of glucagon-like peptide 1 (GLP-1). We have shown that intestinal L cells express VGLUT2, a VGLUT isoform, which suggests that L cells secrete L-glutamate. In the present study, we investigated this possibility using GLUTag mouse clonal L cells. RT-PCR and northern blot analyses revealed expression of the VGLUT1 and VGLUT2 genes, but not of the VGLUT3 gene. Western blot analysis revealed immunological counterparts for VGLUT2, whereas an immunological counterpart of VGLUT1 was not detected. Indirect immunofluorescence microscopy revealed a punctate distribution of VGLUT2 immunoreactivity throughout the cells, which co-localized with GLP-1. Double-labeling immunoelectronmicroscopy confirmed the association of VGLUT2 with GLP-1-containing secretory granules. The membrane fraction exhibited ATP-dependent L-glutamate uptake, which was sensitive to bafilomycin A1 (a vacuolar proton ATPase inhibitor) and Evans blue (a VGLUT inhibitor) but insensitive to D,L-aspartate. Upon depolarization with KCl, GLUTag cells secreted appreciable amounts of L-glutamate and GLP-1. D-Glucose and methyl-alpha-D-glucopyranoside, stimulators of exocytosis of GLP-1, also triggered the secretion of L-glutamate. The L-glutamate secretion was partially dependent on Ca2+ and sensitive to bafilomycin A1. These results demonstrated that GLUTag cells stored L-glutamate in secretory granules and secreted it with GLP-1 by exocytosis. As GLUTag cells and intestinal L cells express kainate receptors and plasma membrane glutamate transporters, these results support the concept of L-glutamate-mediated intercellular signaling in the vicinity of intestinal L cells.

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