Vesicular nucleotide transporter is a molecular target of eicosapentaenoic acid for neuropathic and inflammatory pain treatment.

Eicosapentaenoic acid (EPA), an omega-3 (ω-3) polyunsaturated fatty acid, is an essential nutrient that exhibits antiinflammatory, neuroprotective, and cardiovascular-protective activities. Although EPA is used as a nutrient-based pharmaceutical agent or dietary supplement, its molecular target(s) is debatable. Here, we showed that EPA and its metabolites strongly and reversibly inhibit vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and release of adenosine triphosphate (ATP) in purinergic chemical transmission. In vitro analysis showed that EPA inhibits human VNUT-mediated ATP uptake at a half-maximal inhibitory concentration (IC50) of 67 nM, acting as an allosteric modulator through competition with Cl-. EPA impaired vesicular ATP release from neurons without affecting the vesicular release of other neurotransmitters. In vivo, VNUT-/- mice showed a delay in the onset of neuropathic pain and resistance to both neuropathic and inflammatory pain. EPA potently attenuated neuropathic and inflammatory pain in wild-type mice but not in VNUT-/- mice without affecting the basal nociception. The analgesic effect of EPA was canceled by the intrathecal injection of purinoceptor agonists and was stronger than that of existing drugs used for neuropathic pain treatment, with few side effects. Neuropathic pain impaired insulin sensitivity in previous studies, which was improved by EPA in the wild-type mice but not in the VNUT-/- mice. Our results showed that VNUT is a molecular target of EPA that attenuates neuropathic and inflammatory pain and insulin resistance. EPA may represent a unique nutrient-based treatment and prevention strategy for neurological, immunological, and metabolic diseases by targeting purinergic chemical transmission.

Vesicular nucleotide transporter mediates ATP release and migration in neutrophils.

Neutrophils migrate to sites infected by pathogenic microorganisms. This migration is regulated by neutrophil-secreted ATP, which stimulates neutrophils in an autocrine manner through purinergic receptors on the plasma membrane. Although previous studies have shown that ATP is released through channels at the plasma membrane of the neutrophil, it remains unknown whether it is also released through alternate secretory systems involving vesicular mechanisms. In this study, we investigated the possible involvement of vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and nucleotide release, in ATP secretion from neutrophils. RT-PCR and Western blotting analysis indicated that VNUT is expressed in mouse neutrophils. Immunohistochemical analysis indicated that VNUT mainly colocalized with matrix metalloproteinase-9 (MMP-9), a marker of tertiary granules, which are secretory organelles. In mouse neutrophils, ATP release was inhibited by clodronate, which is a potent VNUT inhibitor. Furthermore, neutrophils from VNUT-/- mice did not release ATP and exhibited significantly reduced migration in vitro and in vivo These findings suggest that tertiary granule-localized VNUT is responsible for vesicular ATP release and subsequent neutrophil migration. Thus, these findings suggest an additional mechanism through which ATP is released by neutrophils.

Reducing phosphorus accumulation in rice grains with an impaired transporter in the node.

Phosphorus is an important nutrient for crop productivity. More than 60% of the total phosphorus in cereal crops is finally allocated into the grains and is therefore removed at harvest. This removal accounts for 85% of the phosphorus fertilizers applied to the field each year. However, because humans and non-ruminants such as poultry, swine and fish cannot digest phytate, the major form of phosphorus in the grains, the excreted phosphorus causes eutrophication of waterways. A reduction in phosphorus accumulation in the grain would contribute to sustainable and environmentally friendly agriculture. Here we describe a rice transporter, SULTR-like phosphorus distribution transporter (SPDT), that controls the allocation of phosphorus to the grain. SPDT is expressed in the xylem region of both enlarged- and diffuse-vascular bundles of the nodes, and encodes a plasma-membrane-localized transporter for phosphorus. Knockout of this gene in rice (Oryza sativa) altered the distribution of phosphorus, with decreased phosphorus in the grains but increased levels in the leaves. Total phosphorus and phytate in the brown de-husked rice were 20-30% lower in the knockout lines, whereas yield, seed germination and seedling vigour were not affected. These results indicate that SPDT functions in the rice node as a switch to allocate phosphorus preferentially to the grains. This finding provides a potential strategy to reduce the removal of phosphorus from the field and lower the risk of eutrophication of waterways.

AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis.

Ascorbate is an antioxidant and coenzyme for various metabolic reactions in vivo. In plant chloroplasts, high ascorbate levels are required to overcome photoinhibition caused by strong light. However, ascorbate is synthesized in the mitochondria and the molecular mechanisms underlying ascorbate transport into chloroplasts are unknown. Here we show that AtPHT4;4, a member of the phosphate transporter 4 family of Arabidopsis thaliana, functions as an ascorbate transporter. In vitro analysis shows that proteoliposomes containing the purified AtPHT4;4 protein exhibit membrane potential- and Cl(-)-dependent ascorbate uptake. The AtPHT4;4 protein is abundantly expressed in the chloroplast envelope membrane. Knockout of AtPHT4;4 results in decreased levels of the reduced form of ascorbate in the leaves and the heat dissipation process of excessive energy during photosynthesis is compromised. Taken together, these observations indicate that the AtPHT4;4 protein is an ascorbate transporter at the chloroplast envelope membrane, which may be required for tolerance to strong light stress.

Inhibitors of ATP release inhibit vesicular nucleotide transporter.

Vesicular nucleotide transporter (VNUT) is responsible for vesicular ATP storage in ATP-secreting cells. In the present study, we examined the effects on VNUT-mediated transport of ATP release inhibitors such as ATP-binding cassette (ABC) proteins, hemichannels, maxi anion channels and P2X7 receptor. The ATP transport activity of proteoliposomes containing purified human VNUT was blocked by glibenclamide, carbenoxolone, 18 α-glycyrrhetinic acid, flufenamic acid, arachidonic acid and A438079 without the formation of Δψ (positive inside) as a driving force being affected. Thus, inhibitors of ATP release may inhibit VNUT and subsequent ATP release, since the previous works proved that inhibitors of ATP release blocked VNUT-mediated ATP release at the cell level.

Functional characterization of vesicular excitatory amino acid transport by human sialin.

Sialin, the protein coded by SLC17A5, is responsible for membrane potential (Δψ)-driven aspartate and glutamate transport into synaptic vesicles in addition to H+/sialic acid co-transport in lysosomes. Rodent sialin mutants harboring the mutations associated with Salla disease in humans did not transport aspartate and glutamate whereas H+/sialic acid co-transport activity was about one-third of the wild-type protein. In this study, we investigate the effects of various mutations on the transport activities of human sialin. Proteoliposomes containing purified heterologously expressed human sialin exhibited both Δψ-driven aspartate and glutamate transport activity and H+/sialic acid co-transport activity. Aspartate and glutamate transport was not detected in the R39C and K136E mutant forms of SLC17A5 protein associated with Salla disease, whereas H+/sialic acid co-transport activity corresponded to 30-50% of the recombinant wild-type protein. In contrast, SLC17A5 protein harboring the mutations associated with infantile sialic acid storage disease, H183R and Δ268SSLRN272 still showed normal levels of Δψ-driven aspartate and glutamate transport even though H+/sialic acid co-transport activity was absent. Human sialin carrying the G328E mutation that causes both phenotypes, and P334R and G378V mutations that cause infantile sialic acid storage disease showed no transport activity. These results support the idea that people suffering from Salla disease have been defective in aspartergic and glutamatergic neurotransmissions.