Polyamine release and vesicular polyamine transporter expression in megakaryoblastic cells and platelets.

Polyamines not only play essential roles in cell growth and function of living organisms but are also released into the extracellular space and function as regulators of chemical transduction, although the cells from which they are released and their mode of release are not well understood. The vesicular polyamine transporter (VPAT), encoded by the SLC18B1 is responsible for the vesicular storage of spermine and spermidine, followed by their vesicular release from secretory cells. Focusing on VPAT will help identify polyamine-secreting cells and new polyamine functions. In this study, we investigated the possible involvement of VPAT in vesicular release of polyamines in MEG-01 clonal megakaryoblastic cells and platelets. RT-PCR, western blotting, and immunohistochemistry revealed VPAT expression in MEG-01 cells. MEG-01 cells secreted polyamines upon A23187 stimulation in the presence of Ca2+, which is temperature-dependent and sensitive to bafilomycin A1. A23187-induced polyamine secretion from MEG-01 cells was reduced by treatment with reserpine, VPAT inhibitors, or VPAT RNA interference. Platelets also expressed VPAT, displaying a punctate distribution, and released spermidine upon A23187 and thrombin stimulation. These findings have demonstrated VPAT-mediated vesicular polyamine release from MEG-01 cells, suggesting the presence of similar vesicular polyamine release mechanisms in platelets.

Positive Regulation of S-Adenosylmethionine on Chondrocytic Differentiation via Stimulation of Polyamine Production and the Gene Expression of Chondrogenic Differentiation Factors.

S-adenosylmethionine (SAM) is considered to be a useful therapeutic agent for degenerative cartilage diseases, although its mechanism is not clear. We previously found that polyamines stimulate the expression of differentiated phenotype of chondrocytes. We also found that the cellular communication network factor 2 (CCN2) played a huge role in the proliferation and differentiation of chondrocytes. Therefore, we hypothesized that polyamines and CCN2 could be involved in the chondroprotective action of SAM. In this study, we initially found that exogenous SAM enhanced proteoglycan production but not cell proliferation in human chondrocyte-like cell line-2/8 (HCS-2/8) cells. Moreover, SAM enhanced gene expression of cartilage-specific matrix (aggrecan and type II collagen), Sry-Box transcription factor 9 (SOX9), CCN2, and chondroitin sulfate biosynthetic enzymes. The blockade of the methionine adenosyltransferase 2A (MAT2A) enzyme catalyzing intracellular SAM biosynthesis restrained the effect of SAM on chondrocytes. The polyamine level in chondrocytes was higher in SAM-treated culture than control culture. Additionally, Alcian blue staining and RT-qPCR indicated that the effects of SAM on the production and gene expression of aggrecan were reduced by the inhibition of polyamine synthesis. These results suggest that the stimulation of polyamine synthesis and gene expression of chondrogenic differentiation factors, such as CCN2, account for the mechanism underlying the action of SAM on chondrocytes.

Functional characterization and tissue localization of the facilitative glucose transporter GLUT12.

Facilitative glucose transporters (GLUTs) play crucial roles in glucose utilization and homeostasis. GLUT12 was initially isolated as a novel GLUT4-like transporter involved in insulin-dependent glucose transport. However, tissue distribution and biochemical properties of GLUT12 are not well understood. In this study, we investigated the basic kinetic properties and tissue distribution of GLUT12. Human GLUT12 and GLUT1 were overexpressed and purified using Ni-NTA column chromatography. Reconstituted proteoliposomes showed time-dependent d-glucose transport activity, which was inhibited by phloretin and dehydroascorbate. Dose dependence of glucose transport revealed a KM and Vmax values of 6.4 mM and 1.2 µmol/mg/min, respectively, indicating that GLUT12 is a high-affinity type GLUT. Glucose transport by GLUT12 was inhibited by ATP and glucose-1-phosphate, glucose-6-phosphate and disaccharides (properties similar to those of GLUT1). Indirect immunohistochemistry revealed the distribution of mouse GLUT12 in the apical region of distal tubules and collecting ducts in the kidney and epithelial cells of the jejunum. In addition to these cells, GLUT12 was present in chromaffin cells in the adrenal medulla, the anterior pituitary lobe, as well as the thyroid and pyloric glands. These tissue distributions suggest a unique function of GLUT12, besides that of an insulin-dependent glucose transport.

The mitochondrial inner membrane protein LETM1 modulates cristae organization through its LETM domain.

LETM1 is a mitochondrial inner membrane protein that is required for maintaining the mitochondrial morphology and cristae structures, and regulates mitochondrial ion homeostasis. Here we report a role of LETM1 in the organization of cristae structures. We identified four amino acid residues of human LETM1 that are crucial for complementation of the growth deficiency caused by gene deletion of a yeast LETM1 orthologue. Substituting amino acid residues with alanine disrupts the correct assembly of a protein complex containing LETM1 and prevents changes in the mitochondrial morphology induced by exogenous LETM1 expression. Moreover, the LETM1 protein changes the shapes of the membranes of in vitro-reconstituted proteoliposomes, leading to the formation of invaginated membrane structures on artificial liposomes. LETM1 mutant proteins with alanine substitutions fail to facilitate the formation of invaginated membrane structures, suggesting that LETM1 plays a fundamental role in the organization of mitochondrial membrane morphology.

Function of essential chloride and arginine residue in nucleotide binding to vesicular nucleotide transporter.

Vesicular nucleotide transporter (VNUT) plays a key role in purinergic signalling through its ability to transport nucleotides. VNUT belongs to the SLC17 family, which includes vesicular glutamate transporters (VGLUTs) and Type I Na+/phosphate cotransporters. All of these transporters exhibit membrane potential and Cl--dependent organic anion transport activity and have essential arginine in the transmembrane region. Previously, we reported that ketoacids inhibit these transporters through modulation of Cl- activation. Although this regulation is important to control signal transmission, the mechanisms underlying Cl--dependent regulation are unclear. Here, we examined the functional roles of Cl- and essential arginine residue on ATP binding to VNUT using the fluorescent ATP analogue trinitrophenyl-ATP (TNP-ATP). The fluorescence of TNP-ATP was enhanced by VNUT, whereas no enhancement was observed by VGLUT. Concentration-dependence curves showed that TNP-ATP was a high-affinity fluorescent probe for VNUT, with a Kd of 4.8 µM. TNP-ATP binding was competitive to ATP and showed similar specificity to transport activity. Addition of Cl- and ketoacids did not affect the apparent affinity for TNP-ATP. The Arg119 to Ala mutant retained TNP-ATP binding ability with slightly reduced affinity. Overall, these results indicated that Cl- and essential arginine were not important for ATP binding.

Outward open conformation of a Major Facilitator Superfamily multidrug/H+ antiporter provides insights into switching mechanism.

Multidrug resistance (MDR) poses a major challenge to medicine. A principle cause of MDR is through active efflux by MDR transporters situated in the bacterial membrane. Here we present the crystal structure of the major facilitator superfamily (MFS) drug/H+ antiporter MdfA from Escherichia coli in an outward open conformation. Comparison with the inward facing (drug binding) state shows that, in addition to the expected change in relative orientations of the N- and C-terminal lobes of the antiporter, the conformation of TM5 is kinked and twisted. In vitro reconstitution experiments demonstrate the importance of selected residues for transport and molecular dynamics simulations are used to gain insights into antiporter switching. With the availability of structures of alternative conformational states, we anticipate that MdfA will serve as a model system for understanding drug efflux in MFS MDR antiporters.

Purification and reconstitution of polyspecific H+/organic cation antiporter human MATE1.

Human MATE1 (multidrug and toxin extrusion 1, hMATE1) is a H+/organic cation (OC) exchanger responsible for the final step of toxic organic cation excretion in the kidney and liver. To investigate the mechanism of transport, we have established an in vitro assay procedure that includes its expression in insect cells, solubilization with octyl glucoside, purification, and reconstitution into liposomes. The resultant proteoliposomes containing hMATE1 as the sole protein component took up radiolabeled tetraethylammonium (TEA) in a ∆pH-dependent and electroneutral fashion. Furthermore, lipid-detergent micelle containing hMATE1 showed ∆pH-dependent TEA binding similar to transport. Mutated hMATE1 with replacement E273Q completely lacked these TEA binding and transport. In the case of divalent substrates, transport was electrogenic. These observations indicate that the stoichiometry of OC/H+ exchange is independent of substrate charge. Purification and reconstitution of hMATE1 is considered to be suitable for understanding the detailed molecular mechanisms of hMATE1. The results suggest that Glu273 of hMATE1 plays essential roles in substrate binding and transport.

Efficient Mass Spectral Analysis of Active Transporters Overexpressed in Escherichia coli.

Structural analysis of purified active membrane proteins can be performed by mass spectrometry (MS). However, no large-scale expression systems for active eukaryotic membrane proteins are available. Moreover, because membrane proteins cannot easily be digested by trypsin and ionized, they are difficult to analyze by MS. We developed a method for mass spectral analysis of eukaryotic membrane proteins combined with an overexpression system in Escherichia coli. Vesicular glutamate transporter 2 (VGLUT2/SLC17A6) with a soluble α-helical protein and histidine tag on the N- and C-terminus, respectively, was overexpressed in E. coli, solubilized with detergent, and purified by Ni-NTA affinity chromatography. Proteoliposomes containing VGLUT2 retained glutamate transport activity. For MS analysis, the detergent was removed from purified VGLUT2 by trichloroacetic acid precipitation, and VGLUT2 was then subjected to reductive alkylation and tryptic digestion. The resulting peptides were detected with 88% coverage by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS with or without liquid chromatography. Vesicular excitatory amino acid transporter and vesicular acetylcholine transporter were also detected with similar coverage by the same method. Thus this methodology could be used to analyze purified eukaryotic active transporters. Structural analysis with chemical modifiers by MS could have applications in functional binding analysis for drug discovery.

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.

Reconstitution and Transport Analysis of Eukaryotic Transporters in the Post-Genomic Era.

Measuring transport activity through reconstituted proteoliposomes is a key technique to resolve numerous problems found in the traditional methods. The system includes overexpression, purification, and reconstitution of transporters. Mixing of purified transporter with lipid and dilution below the critical micelle concentration result in rapid generation of proteoliposomes. Incubation of proteoliposomes in the presence of a driving force initiates substrate uptake. After starting the reaction, samples are passed through a gel filtration column to separate proteoliposomes from the reaction mixture. Here, we describe step-by-step procedures for such reconstitution assays.

Identification of a vesicular ATP release inhibitor for the treatment of neuropathic and inflammatory pain.

Despite the high incidence of neuropathic and inflammatory pain worldwide, effective drugs with few side effects are currently unavailable for the treatment of chronic pain. Recently, researchers have proposed that inhibitors of purinergic chemical transmission, which plays a key role in the pathological pain response, may allow for targeted treatment of pathological neuropathic and inflammatory pain. However, such therapeutic analgesic agents have yet to be developed. In the present study, we demonstrated that clodronate, a first-generation bisphosphonate with comparatively fewer side effects than traditional treatments, significantly attenuates neuropathic and inflammatory pain unrelated to bone abnormalities via inhibition of vesicular nucleotide transporter (VNUT), a key molecule for the initiation of purinergic chemical transmission. In vitro analyses indicated that clodronate inhibits VNUT at a half-maximal inhibitory concentration of 15.6 nM without affecting other vesicular neurotransmitter transporters, acting as an allosteric modulator through competition with Cl- A low concentration of clodronate impaired vesicular ATP release from neurons, microglia, and immune cells. In vivo analyses revealed that clodronate is more effective than other therapeutic agents in attenuating neuropathic and inflammatory pain, as well as the accompanying inflammation, in wild-type but not VNUT -/- mice, without affecting basal nociception. These findings indicate that clodronate may represent a unique treatment strategy for chronic neuropathic and inflammatory pain via inhibition of vesicular ATP release.

Vesicular nucleotide transporter (VNUT): appearance of an actress on the stage of purinergic signaling.

Vesicular storage of ATP is one of the processes initiating purinergic chemical transmission. Although an active transport mechanism was postulated to be involved in the processes, a transporter(s) responsible for the vesicular storage of ATP remained unidentified for some time. In 2008, SLC17A9, the last identified member of the solute carrier 17 type I inorganic phosphate transporter family, was found to encode the vesicular nucleotide transporter (VNUT) that is responsible for the vesicular storage of ATP. VNUT transports various nucleotides in a membrane potential-dependent fashion and is expressed in the various ATP-secreting cells. Mice with knockout of the VNUT gene lose vesicular storage and release of ATP from neurons and neuroendocrine cells, resulting in blockage of the initiation of purinergic chemical transmission. Thus, VNUT plays an essential role in the vesicular storage and release of ATP. The VNUT knockout mice exhibit resistance for neuropathic pain and a therapeutic effect against diabetes by way of increased insulin sensitivity. Thus, VNUT inhibitors and suppression of VNUT gene expression may be used for therapeutic purposes through suppression of purinergic chemical transmission. This review summarizes the studies to date on VNUT and discusses what we have learned about the relevance of vesicular ATP release as a potential drug target.

Function and expression of a splicing variant of vesicular glutamate transporter 1.

Vesicular glutamate transporter (VGLUT) is an active transporter responsible for vesicular storage of glutamate in synaptic vesicles and plays an essential role in glutamatergic neurotransmission. VGLUT consists of three isoforms, VGLUT1, VGLUT2, and VGLUT3. The VGLUT1 variant, VGLUT1v, with an additional 75-base pair sequence derived from a second intron between exons 2 and 3, which corresponds to 25 amino acid residues in the 1st loop of VGLUT1, is the only splicing variant among VGLUTs, although whether VGLUT1v protein is actually translated at the protein level remains unknown. In the present study, VGLUT1v was expressed in insect cells, solubilized, purified to near homogeneity, and its transport activity was examined. Proteoliposomes containing purified VGLUT1v were shown to accumulate glutamate upon imposition of an inside-positive membrane potential (Δψ). The Δψ-driven glutamate uptake activity requires Cl- and its pharmacological profile and kinetics are comparable to those of other VGLUTs. The retinal membrane contained two VGLUT1 moieties with apparent molecular masses of 65 and 57kDa. VGLUT1v-specific antibodies against an inserted 25-amino acid residue sequence identified a 65-kDa immunoreactive polypeptide. Immunohistochemical analysis indicated that VGLUT1v immunoreactivity is present in photoreceptor cells and is associated with synaptic vesicles. VGLUT1v immunoreactivity is also present in pinealocytes, but not in other areas, including the brain. These results indicated that VGLUT1v exists in a functional state in rat photosensitive cells and is involved in glutamatergic chemical transmission.

Vesicular Polyamine Transporter Mediates Vesicular Storage and Release of Polyamine from Mast Cells.

Mast cells are secretory cells that play an important role in host defense by discharging various intragranular contents, such as histamine and serotonin, upon stimulation of Fc receptors. The granules also contain spermine and spermidine, which can act as modulators of mast cell function, although the mechanism underlying vesicular storage remains unknown. Vesicular polyamine transporter (VPAT), the fourth member of the SLC18 transporter family, is an active transporter responsible for vesicular storage of spermine and spermidine in neurons. In the present study, we investigated whether VPAT functions in mast cells. RT-PCR and Western blotting indicated VPAT expression in murine bone marrow-derived mast cells (BMMCs). Immunohistochemical analysis indicated that VPAT is colocalized with VAMP3 but not with histamine, serotonin, cathepsin D, VAMP2, or VAMP7. Membrane vesicles from BMMCs accumulated spermidine upon the addition of ATP in a reserpine- and bafilomycin A1-sensitive manner. BMMCs secreted spermine and spermidine upon the addition of either antigen or A23187 in the presence of Ca2+, and the antigen-mediated release, which was shown to be temperature-dependent and sensitive to bafilomycin A1 and tetanus toxin, was significantly suppressed by VPAT gene RNA interference. Under these conditions, expression of vesicular monoamine transporter 2 was unaffected, but antigen-dependent histamine release was significantly suppressed, which was recovered by the addition of 1 mm spermine. These results strongly suggest that VPAT is expressed and is responsible for vesicular storage of spermine and spermidine in novel secretory granules that differ from histamine- and serotonin-containing granules and is involved in vesicular release of these polyamines from mast cells.

Structure, Function, and Drug Interactions of Neurotransmitter Transporters in the Postgenomic Era.

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.

Wide expression of type I Na+-phosphate cotransporter 3 (NPT3/SLC17A2), a membrane potential-driven organic anion transporter.

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.

Plasmodium falciparum chloroquine resistance transporter is a H+-coupled polyspecific nutrient and drug exporter.

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.

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.

Identification of a mammalian vesicular polyamine transporter.

Spermine and spermidine act as neuromodulators upon binding to the extracellular site(s) of various ionotropic receptors, such as N-methyl-d-aspartate receptors. To gain access to the receptors, polyamines synthesized in neurons and astrocytes are stored in secretory vesicles and released upon depolarization. Although vesicular storage is mediated in an ATP-dependent, reserpine-sensitive fashion, the transporter responsible for this process remains unknown. SLC18B1 is the fourth member of the SLC18 transporter family, which includes vesicular monoamine transporters and vesicular acetylcholine transporter. Proteoliposomes containing purified human SLC18B1 protein actively transport spermine and spermidine by exchange of H(+). SLC18B1 protein is predominantly expressed in the hippocampus and is associated with vesicles in astrocytes. SLC18B1 gene knockdown decreased both SLC18B1 protein and spermine/spermidine contents in astrocytes. These results indicated that SLC18B1 encodes a vesicular polyamine transporter (VPAT).

Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity.

Neuroendocrine cells store ATP in secretory granules and release it along with hormones that may trigger a variety of cellular responses in a process called purinergic chemical transmission. Although the vesicular nucleotide transporter (VNUT) has been shown to be involved in vesicular storage and release of ATP, its physiological relevance in vivo is far less well understood. In Vnut knockout (Vnut(-/-)) mice, we found that the loss of functional VNUT in adrenal chromaffin granules and insulin granules in the islets of Langerhans led to several significant effects. Vesicular ATP accumulation and depolarization-dependent ATP release were absent in the chromaffin granules of Vnut(-/-) mice. Glucose-responsive ATP release was also absent in pancreatic ß-cells in Vnut(-/-) mice, while glucose-responsive insulin secretion was enhanced to a greater extent than that in wild-type tissue. Vnut(-/-) mice exhibited improved glucose tolerance and low blood glucose upon fasting due to increased insulin sensitivity. These results demonstrated an essential role of VNUT in vesicular storage and release of ATP in neuroendocrine cells in vivo and suggest that vesicular ATP and/or its degradation products act as feedback regulators in catecholamine and insulin secretion, thereby regulating blood glucose homeostasis.