Identification of SLC38A7 (SNAT7) protein as a glutamine transporter expressed in neurons

The SLC38 family of transporters has in total 11 members in humans and they encode amino acid transporters called sodium-coupled amino acid transporters (SNAT). To date, five SNATs have been characterized and functionally subdivided into systems A (SLC38A1, SLC38A2, and SLC38A4) and N (SLC38A3 and SLC38A5) showing the highest transport for glutamine and alanine. Here we present identification of a novel glutamine transporter encoded by the Slc38a7 gene, which we propose should be named SNAT7. This transporter has L-glutamine as the preferred substrate but also transports other amino acids with polar side chains, as well as L-histidine and L-alanine. The expression pattern and substrate profile for SLC38A7 shows highest similarity to the known system N transporters. Therefore, we propose that SLC38A7 is a novel member of this system. We used in situ hybridization and immunohistochemistry with a custom-made antibody to show that SLC38A7 is expressed in all neurons, but not in astrocytes, in the mouse brain. SLC38A7 is unique in being the first system N transporter expressed in GABAergic and also other neurons. The preferred substrate and axonal localization of SLC38A7 close to the synaptic cleft indicates that SLC38A7 could have an important function for the reuptake and recycling of glutamate.     

Functional roles of microglia in the central nervous system

Microglia, a type of perineuronal glial cells in the central nervous system, have been suggested to play various important roles in normal and pathologic brains. In this article, first, we described the association or roles of activated microglia in injury and various brain diseases, and subsequently, summarized microglia-derived physiologically active molecules which will affect the neuronal survival and neuronal growth, and glial function, and finally, discussed the molecular mechanism of microglial activation.     

The contribution of SNAT1 to system A amino acid transporter activity in human placental trophoblast

System A-mediated amino acid transport across the placenta is important for the supply of neutral amino acids needed for fetal growth. All three system A subtypes (SNAT1, 2, and 4) are expressed in human placental trophoblast suggesting there is an important biological role for each. Placental system A activity increases as pregnancy progresses, coinciding with increased fetal nutrient demands. We have previously shown SNAT4-mediated system A activity is higher in first trimester than at term, suggesting that SNAT1 and/or SNAT2 are responsible for the increased system A activity later in gestation. However, the relative contribution of each subtype to transporter activity in trophoblast at term has yet to be evaluated. The purpose of this study was to identify the predominant subtype of system A in cytotrophoblast cells isolated from term placenta, maintained in culture for 66 h, by: (1) measuring mRNA expression of the three subtypes and determining the Michaelis-Menten constants for uptake of the system A-specific substrate, ¹⁴C-MeAIB, (2) investigating the contribution of SNAT1 to total system A activity using siRNA. Results: mRNA expression was highest for the SNAT1 subtype of system A. Kinetic analysis of ¹⁴C-MeAIB uptake revealed two distinct transport systems; system 1: K_m = 0.38 ± 0.12 mM, V_max = 27.8 ± 9.0 pmol/mg protein/20 min, which resembles that reported for SNAT1 and SNAT2 in other cell types, and system 2: K_m = 45.4 ± 25.0 mM, V_max = 1190 ± 291 pmol/mg protein/20 min, which potentially represents SNAT4. Successful knockdown of SNAT1 mRNA using target-specific siRNA significantly reduced system A activity (median 75% knockdown, n = 7). Conclusion: These data enhance our limited understanding of the relative importance of the system A subtypes for amino acid transport in human placental trophoblast by demonstrating that SNAT1 is a key contributor to system A activity at term.     

Zip14 is a complex broad-scope metal-ion transporter whose functional properties support roles in the cellular uptake of zinc and nontransferrin-bound iron

Recent studies have shown that overexpression of the transmembrane protein Zrt- and Irt-like protein 14 (Zip14) stimulates the cellular uptake of zinc and nontransferrin-bound iron (NTBI). Here, we directly tested the hypothesis that Zip14 transports free zinc, iron, and other metal ions by using the Xenopus laevis oocyte heterologous expression system, and use of this approach also allowed us to characterize the functional properties of Zip14. Expression of mouse Zip14 in RNA-injected oocytes stimulated the uptake of (55)Fe in the presence of l-ascorbate but not nitrilotriacetic acid, indicating that Zip14 is an iron transporter specific for ferrous ion (Fe(2+)) over ferric ion (Fe(3+)). Zip14-mediated (55)Fe(2+) uptake was saturable (K(0.5) ≈ 2 μM), temperature-dependent (apparent activation energy, E(a) = 15 kcal/mol), pH-sensitive, Ca(2+)-dependent, and inhibited by Co(2+), Mn(2+), and Zn(2+). HCO(3)(-) stimulated (55)Fe(2+) transport. These properties are in close agreement with those of NTBI uptake in the perfused rat liver and in isolated hepatocytes reported in the literature. Zip14 also mediated the uptake of (109)Cd(2+), (54)Mn(2+), and (65)Zn(2+) but not (64)Cu (I or II). (65)Zn(2+) uptake also was saturable (K(0.5) ≈ 2 μM) but, notably, the metal-ion inhibition profile and Ca(2+) dependence of Zn(2+) transport differed from those of Fe(2+) transport, and we propose a model to account for these observations. Our data reveal that Zip14 is a complex, broad-scope metal-ion transporter. Whereas zinc appears to be a preferred substrate under normal conditions, we found that Zip14 is capable of mediating cellular uptake of NTBI characteristic of iron-overload conditions.     

SNAT4 isoform of system A amino acid transporter is expressed in human placenta

The system A amino acid transporter is encoded by three members of the Slc38 gene family, giving rise to three subtypes: Na+-coupled neutral amino acid transporter (SNAT)1, SNAT2, and SNAT4. SNAT2 is expressed ubiquitously in mammalian tissues; SNAT1 is predominantly expressed in heart, brain, and placenta; and SNAT4 is reported to be expressed solely by the liver. In the placenta, system A has an essential role in the supply of neutral amino acids needed for fetal growth. In the present study, we examined expression and localization of SNAT1, SNAT2, and SNAT4 in human placenta during gestation. Real-time quantitative PCR was used to examine steady-state levels of system A subtype mRNA in early (6-10 wk) and late (10-13 wk) first-trimester and full-term (38-40 wk) placentas. We detected mRNA for all three isoforms from early gestation onward. There were no differences in SNAT1 and SNAT2 mRNA expression with gestation. However, SNAT4 mRNA expression was significantly higher early in the first trimester compared with the full-term placenta (P < 0.01). We next investigated SNAT4 protein expression in human placenta. In contrast to the observation for gene expression, Western blot analysis revealed that SNAT4 protein expression was significantly higher at term compared with the first trimester (P < 0.05). Immunohistochemistry and Western blot analysis showed that SNAT4 is localized to the microvillous and basal plasma membranes of the syncytiotrophoblast, suggesting a role for this isoform of system A in amino acid transport across the placenta. This study therefore provides the first evidence of SNAT4 mRNA and protein expression in the human placenta, both at the first trimester and at full term.     

ATP binding cassette transporter A1 - key roles in cellular lipid transport and atherosclerosis

ATP-binding cassette transporter A1 (ABCA1) was recently recognized as the mutant molecule responsible for Tangier disease with low HDL levels, accumulation of cholesteryl esters in tissues, and increased risk of cardiovascular disease. Extensive studies for the past 2 years have recognized the critical role of ABCA1 in cholesterol and phospholipid trafficking. Since the removal of cholesterol from tissues is a key step in the prevention of atherosclerosis, significant attention has been focused on this molecule. Natural ABCA1 mutations in Tangier disease (TD) patients and WHAM chickens together with induced mutation in ABCA1 knock-out mice unequivocally established the important role of ABCA1 in maintaining circulating HDL levels and promoting cholesterol efflux from the arterial wall. Mice lacking ABCA1 showed similar phenotypes observed in Tangier disease patients with low levels of HDL. Further understanding of the roles of ABCA1 in lipid transport and atherosclerosis became clear from studies with ABCA1 transgenic mice. These mice showed enhanced cholesterol efflux from macrophages and reduced atherosclerotic lesion formation. The promoter of the ABCA1 gene has been mapped to a large extent, with the exception of cAMP response element. The present review summarizes recent developments on the role of ABCA1 in cholesterol efflux and prevention of atherosclerosis. Given the antiatherogenic properties of ABCA1, this molecule can serve as an appropriate target for developing drugs to treat individuals with low levels of HDL.     

Functional roles for myosin 1c in cellular signaling pathways

Cellular signaling pathways underlie the transfer of information throughout the cell and to adjoining cells and so govern most critical cellular functions. Increasing evidence points to the molecular motor myosin 1c as a prominent player in many signaling cascades, from the integrin-dependent signaling involved in cell migration to the signaling events underlying insulin resistance. Myosin 1c functions on these pathways both via an important role in regulating lipid raft recycling and also via direct involvement in signaling cascades. This review provides an overview of the functional involvement of myosin 1c in cellular signaling and discusses the possible potential for myosin 1c as a target for drug-based treatments for human diseases.     

Heterologous expression of the glutamine transporter SNAT3 in Xenopus oocytes is associated with four modes of uncoupled transport

The glutamine transporter SNAT3 (SLC38A3, former SN1) plays a major role in glutamine release from brain astrocytes and in glutamine uptake into hepatocytes and kidney epithelial cells. Here we expressed rat SNAT3 in oocytes of Xenopus laevis and reinvestigated its transport modes using two-electrode voltage clamp and pH-sensitive microelectrodes. In addition to the established coupled Na+-glutamine-cotransport/H+ antiport, we found that there are three conductances associated with SNAT3, two dependent and one independent of the amino acid substrate. The glutamine-dependent conductance is carried by cations at pH 7.4, whereas at pH 8.4 the inward currents are still dependent on the presence of external Na+ but are carried by H+. Mutation of threonine 380 to alanine abolishes the cation conductance but leaves the proton conductance intact. Under Na+-free conditions, where the substrate-dependent conductance is suppressed, a substrate-independent, outwardly rectifying current becomes apparent at pH 8.4 that is carried by K+ and H+. In addition, we identified a glutamine-dependent uncoupled Na+/H+ exchange activity that becomes apparent upon removal of Na+ in the presence of glutamine. In conclusion, our results suggest that, in addition to coupled transport, SNAT3 mediates four modes of uncoupled ion movement across the membrane.     

The distribution of cholinergic neurons in the human central nervous system

Choline acetyltransferase (ChAT), the enzyme responsible for the biosynthesis of acetylcholine, is presently the most specific marker for identifying cholinergic neurons in the central and peripheral nervous systems. The present article reviews immunohistochemical and in situ hybridization studies on the distribution of neurons expressing ChAT in the human central nervous system. Neurons with both immunoreactivity and in situ hybridization signals of ChAT are observed in the basal forebrain (diagonal band of Broca and nucleus basalis of Meynert), striatum (caudate nucleus, putamen and nucleus accumbens), cerebral cortex, mesopontine tegmental nuclei (pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus and parabigeminal nucleus), cranial motor nuclei and spinal motor neurons. The cerebral cortex displays regional and laminal differences in the distribution of neurons with ChAT. The medial septal nucleus and medial habenular nucleus contain immunoreactive neurons for ChAT, which are devoid of ChAT mRNA signals. This is probably because there is a small number of cholinergic neurons with a low level of ChAT gene expression in these nuclei of human. Possible connections and speculated functions of these neurons are briefly summarized.     

Enzymatic suppression of the membrane conductance associated with the glutamine transporter SNAT3 expressed in Xenopus oocytes by carbonic anhydrase II

The transport activity of the glutamine/neutral amino acid transporter SNAT3 (former SN1, SLC38A3), expressed in oocytes of the frog Xenopus laevis is associated with a non-stoichiometrical membrane conductance selective for Na⁺ and/or H⁺ (Schneider, H.P., S. Bröer, A. Bröer, and J.W. Deitmer. 2007. J. Biol. Chem. 282:3788–3798). When we expressed SNAT3 in frog oocytes, the glutamine-induced membrane conductance was suppressed, when carbonic anhydrase isoform II (CAII) had been injected into the oocytes. Transport of substrate, however, was not affected by CAII. The reduction of the membrane conductance by CAII was dependent on the presence of CO₂/HCO₃ ⁻, and could be reversed by blocking the catalytic activity of CAII by ethoxyzolamide (10 μM). Coexpression of wild-type CAII or a N-terminal CAII mutant with SNAT3 also reduced the SNAT3- associated membrane conductance. The catalytically inactive CAII mutant V143Y coexpressed in oocytes did not affect SNAT3-associated membrane conductance. Our results reveal a new type of interaction between CAII and a transporter-associated cation conductance, and support the hypothesis that the transport of substrate and the non-stoichiometrical ion conductance are independent of each other. This study also emphasizes the importance of carbonic anhydrase activity and the presence of CO₂-bicarbonate buffers for membrane transport processes.     

Functional properties of transfected human DMT1 iron transporter

Recently, mutation of the DMT1 gene has been discovered to cause ineffective intestinal iron uptake and abnormal body iron metabolism in the anemic Belgrade rat and mk mouse. DMT1 transports first-series transition metals, but only iron turns on an inward proton current. The process of iron transport was studied by transfection of human DMT1 into the COS-7 cell line. Native and epitope-tagged human DMT1 led to increased iron uptake. The human gene with the Belgrade rat mutation was found to have one-fifth of the activity of the wild-type protein. The pH optimum of human DMT1 iron uptake was 6.75, which is equivalent to the pH of the duodenal brush border. The transporter demonstrates uptake without saturation from 0 to 50 microM iron, recapitulating earlier studies of isolated intestinal enterocytes. Diethylpyrocarbonate inhibition of iron uptake in DMT1-transfected cells suggests a functional role for histidine residues. Finally, a model is presented that incorporates the selectivity of the DMT1 transporter for transition metals and a potential role for the inward proton current.     

Characterizing unexpected interactions of a glutamine transporter inhibitor with members of the SLC1A transporter family

The solute carrier 1A family comprises a group of membrane proteins that act as dual-function amino acid transporters and chloride (Cl⁻) channels and includes the alanine serine cysteine transporters (ASCTs) as well as the excitatory amino acid transporters. ASCT2 is regarded as a promising target for cancer therapy, as it can transport glutamine and other neutral amino acids into cells and is upregulated in a range of solid tumors. The compound L-γ-glutamyl-p-nitroanilide (GPNA) is widely used in studies probing the role of ASCT2 in cancer biology; however, the mechanism by which GPNA inhibits ASCT2 is not entirely clear. Here, we used electrophysiology and radiolabelled flux assays to demonstrate that GPNA activates the Cl⁻ conductance of ASCT2 to the same extent as a transported substrate, whilst not undergoing the full transport cycle. This is a previously unreported phenomenon for inhibitors of the solute carrier 1A family but corroborates a body of literature suggesting that the structural requirements for transport are distinct from those for Cl⁻ channel formation. We also show that in addition to its currently known targets, GPNA inhibits several of the excitatory amino acid transporters. Together, these findings raise questions about the true mechanisms of its anticancer effects.     

Functional lipidomics: the roles of specialized lipids and lipid-protein interactions in modulating neuronal function

Lipids fulfill multiple specialized roles in neuronal function. In brain, the conduction of electrical impulses, synaptic function, and complex signaling pathways depend on the temporally and spatially coordinated interactions of specialized lipids (e.g., arachidonic acid and plasmalogens), proteins (e.g., ion channels, phospholipases and cyclooxygenases) and integrative lipid-protein interactions. Recent technical advances in mass spectrometry have allowed unparalled insight into the roles of lipids in neuronal function. Through shotgun lipidomics and multidimensional mass spectrometry, in conjunction with the identification of new classes of phospholipases (e.g., calcium dependent and calcium independent intracellular phospholipases), new roles for lipids in cerebral function have been accrued. This review summarizes the advances in our understanding of the types of lipids and phospholipases in the brain and the role of functional lipidomics in increasing our chemical understanding of complex neuronal processes.     

On the roles of colour and scent in a specialized floral mimicry system

Background and Aims

Sexually deceptive orchids achieve cross-pollination by mimicking the mating signals of female insects, generally hymenopterans. This pollination mechanism is often highly specific as it is based primarily on the mimicry of mating signals, especially the female sex pheromones of the targeted pollinator. Like many deceptive orchids, the Mediterranean species Ophrys arachnitiformis shows high levels of floral trait variation, especially in the colour of the perianth, which is either green or white/pinkinsh within populations. The adaptive significance of perianth colour polymorphism and its influence on pollinator visitation rates in sexually deceptive orchids remain obscure.

Methods

The relative importance of floral scent versus perianth colour in pollinator attraction in this orchid pollinator mimicry system was evaluated by performing floral scent analyses by gas chromatography-mass spectrometry (GC-MS) and behavioural bioassays with the pollinators under natural conditions were performed.

Key Results

The relative and absolute amounts of behaviourally active compounds are identical in the two colour morphs of O. arachnitiformis. Neither presence/absence nor the colour of the perianth (green versus white) influence attractiveness of the flowers to Colletes cunicularius males, the main pollinator of O. arachnitiformis.

Conclusion

Chemical signals alone can mediate the interactions in highly specialized mimicry systems. Floral colour polymorphism in O. arachnitiformis is not subjected to selection imposed by C. cunicularius males, and an interplay between different non-adaptive processes may be responsible for the maintenance of floral colour polymorphism both within and among populations.