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The human malaria parasite Plasmodium falciparum relies on lipids to survive; this makes its lipid metabolism an attractive drug target. The lipid phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell membrane (RBC) bilayer; however, some studies suggest that infection with the intracellular parasite results in the presence of this lipid in the RBC membrane outer leaflet, where it could act as a recognition signal to phagocytes. Here, we used fluorescent lipid analogues and probes to investigate the enzymatic reactions responsible for maintaining asymmetry between membrane leaflets, and found that in parasitised RBCs the maintenance of membrane asymmetry was partly disrupted, and PS was increased in the outer leaflet. We examined the underlying causes for the differences between uninfected and infected RBCs using fluorescent dyes and probes, and found that calcium levels increased in the infected RBC cytoplasm, whereas membrane cholesterol was depleted from the erythrocyte plasma membrane. We explored the resulting effect of PS exposure on enhanced phagocytosis by monocytes, and show that infected RBCs must expend energy to limit phagocyte recognition, and provide experimental evidence that PS exposure contributes to phagocytic recognition of P. falciparum-infected RBCs. Together, these findings underscore the pivotal role for PS exposure on the surface of Plasmodium falciparum-infected erythrocytes for in vivo interactions with the host immune system, and provide a rationale for targeted antimalarial drug design.

Dietary protein restriction has beneficial impacts on metabolic health. B0AT1 (SLC6A19) is the major transporter of neutral amino acids at the intestinal epithelia and absorbs the bulk of the diet-derived neutral amino acids from the intestinal lumen. It also reabsorbs neutral amino acids in the renal proximal tubules. Mice lacking B0AT1 show cellular outcomes of protein restriction, such as high FGF21 levels and low mTORC1 activity. Moreover, they have improved glucose homeostasis and resist diet-induced obesity. In this study, we investigated the relationship between protein restriction and dietary protein intake in C57Bl6/J wild-type (wt) and SLC6A19-knockout (SLC6A19ko) mice. When SLC6A19ko mice were fed diets containing 5%, 25%, or 52% of their total calories derived from protein, no differences in food intake or weight gain were observed. All essential amino acids significantly positively correlated with increasing dietary casein content in the wt mice. The SLC6A19ko mice showed reduced postprandial levels of essential amino acids in plasma, particularly following high-protein diets. Upon fasting, essential amino acids were the same in the wt and SLC6A19ko mice due to reduced amino acid catabolism. Bacterial metabolites originating from amino acid fermentation correlated with the dietary protein content, but showed a complex profile in the blood of the SLC6A19ko mice. This study highlights the potential of SLC6A19 as a knock-out or inhibition target to induce protein restriction for the treatment of metabolic disorders.

The small intestine mediates the absorption of amino acids after ingestion of protein and sustains the supply of amino acids to all tissues. The small intestine is an important contributor to plasma amino acid homeostasis, while amino acid transport in the large intestine is more relevant for bacterial metabolites and fluid secretion. A number of rare inherited disorders have contributed to the identification of amino acid transporters in epithelial cells of the small intestine, in particular cystinuria, lysinuric protein intolerance, Hartnup disorder, iminoglycinuria, and dicarboxylic aminoaciduria. These are most readily detected by analysis of urine amino acids, but typically also affect intestinal transport. The genes underlying these disorders have all been identified. The remaining transporters were identified through molecular cloning techniques to the extent that a comprehensive portrait of functional cooperation among transporters of intestinal epithelial cells is now available for both the basolateral and apical membranes. Mouse models of most intestinal transporters illustrate their contribution to amino acid homeostasis and systemic physiology. Intestinal amino acid transport activities can vary between species, but these can now be explained as differences of amino acid transporter distribution along the intestine.

The asymmetric distribution of phospholipids in the plasma/organellar membranes is generated and maintained through phospholipid flippases in resting cells, but becomes disrupted in apoptotic cells and activated platelets, resulting in phosphatidylserine (PS) exposure on the cell surface. Stable PS exposure during apoptosis requires inactivation of flippases to prevent PS from being reinternalized. Here we show that flippase ATP8A1 is highly expressed in both murine and human platelets, but is not present in the plasma membrane. ATP8A1 is cleaved by the cysteine protease calpain during apoptosis, and the cleavage is prevented indirectly by caspase inhibition, involving blockage of calcium influx into platelets and subsequent calpain activation. In contrast, in platelets activated with thrombin and collagen and exposing PS, ATP8A1 remains intact. These data reveal a novel mechanism of flippase cleavage and suggest that flippase activity in intracellular membranes differs between platelets undergoing apoptosis and activation.

Apicomplexan parasites are auxotrophic for a range of amino acids which must be salvaged from their host cells, either through direct uptake or degradation of host proteins. Here, we describe a family of plasma membrane-localized amino acid transporters, termed the Apicomplexan Amino acid Transporters (ApiATs), that are ubiquitous in apicomplexan parasites. Functional characterization of the ApiATs of Toxoplasma gondii indicate that several of these transporters are important for intracellular growth of the tachyzoite stage of the parasite, which is responsible for acute infections. We demonstrate that the ApiAT protein TgApiAT5-3 is an exchanger for aromatic and large neutral amino acids, with particular importance for L-tyrosine scavenging and amino acid homeostasis, and that TgApiAT5-3 is critical for parasite virulence. Our data indicate that T. gondii expresses additional proteins involved in the uptake of aromatic amino acids, and we present a model for the uptake and homeostasis of these amino acids. Our findings identify a family of amino acid transporters in apicomplexans, and highlight the importance of amino acid scavenging for the biology of this important phylum of intracellular parasites. Author summary: The Apicomplexa comprise a large number of parasitic protozoa that have obligate intracellular lifestyles and cause significant human and animal diseases, including malaria, cryptosporidiosis, toxoplasmosis, coccidiosis in poultry, and various cattle fevers. Apicomplexans must scavenge essential nutrients from their hosts in order to proliferate and cause disease, including a range of amino acids. The direct uptake of these nutrients is presumed to be mediated by transporter proteins located in the plasma membrane of intracellular stages, although the identities of these proteins are poorly defined. Using a combination of bioinformatic, genetic, cell biological, and physiological approaches, we have characterized an apicomplexan-specific family of plas

Recent studies have established that dietary protein restriction improves metabolic health and glucose homeostasis. SLC6A19 (B⁰AT1) is the major neutral amino acid transporter in the intestine and carries out the bulk of amino acid absorption from the diet. Mice lacking SLC6A19 show signs of protein restriction, have improved glucose tolerance, and are protected from diet-induced obesity. Pharmacological blockage of this transporter could be used to induce protein restriction and to treat metabolic diseases such as type 2 diabetes. A few novel inhibitors of SLC6A19 have recently been identified using in vitro compound screening, but it remains unclear whether these compounds block the transporter in vivo. To evaluate the efficacy of SLC6A19 inhibitors biomarkers are required that can reliably detect successful inhibition of the transporter in mice. A gas chromatography mass spectrometry (GC-MS)-based untargeted metabolomics approach was used to discriminate global metabolite profiles in plasma, urine and faecal samples from SLC6A19ko and wt mice. Due to inefficient absorption in the intestine and lack of reabsorption in the kidney, significantly elevated amino acids levels were observed in urine and faecal samples. By contrast, a few neutral amino acids were reduced in the plasma of male SLC6A19ko mice as compared to other biological samples. Metabolites of bacterial protein fermentation such as p-cresol glucuronide and 3-indole-propionic acid were more abundant in SLC6A19ko mice, indicating protein malabsorption of dietary amino acids. Consistently, plasma appearance rates of [14C]-labelled neutral amino acids were delayed in SLC6A19ko mice as compared to wt after intra-gastric administration of a mixture of amino acids. Receiver operating characteristic (ROC) curve analysis was used to validate the potential use of these metabolites as biomarkers. These findings provide putative metabolite biomarkers that can be used to detect protein malabsorption and the inhibit

Organization of the plasma membrane into specialized substructures in different blood lineages facilitates important biological functions including proper localization of receptors at the plasma membrane as well as the initiation of crucial intracellular signaling cascades. The eukaryotic plasma membrane is a lipid bilayer that consists of asymmetrically distributed phospholipids. This asymmetry is actively maintained by membrane-embedded lipid transporters, but there is only limited data available about the molecular identity of the predominantly active transporters and their substrate specificity in different leukocyte subsets. We demonstrate here that the P4-type ATPase ATP11C mediates significant flippase activity in all murine leukocyte subsets. Loss of ATP11C resulted in a defective internalization of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in comparison to control cells. The diminished flippase activity caused increased PS exposure on 7-aminoactinomycin D−(7-AAD−) viable pro-B cells freshly isolated from the bone marrow of ATP11C-deficient mice, which was corrected upon a 2-hour resting period in vitro. Despite the impaired flippase activity in all immune cell subsets, the only other blood cell type with an accumulation of PS on the surface were viable 7-AAD− developing T cells but this did not result in any discernable effect on their development in the thymus. These findings show that all leukocyte lineages exhibit flippase activity, and identify ATP11C as an aminophospholipid translocase in immune cells.

A number of different strategies have been used to identify genes for which genetic variation contributes to type 1 diabetes (T1D) pathogenesis. Genetic studies in humans have identified .40 loci that affect the risk for developing T1D, but the underlying causative alleles are often difficult to pinpoint or have subtle biological effects. A complementary strategy to identifying “natural” alleles in the human population is to engineer “artificial” alleles within inbred mouse strains and determine their effect on T1D incidence. We describe the use of the Sleeping Beauty (SB) transposon mutagenesis system in the nonobese diabetic (NOD) mouse strain, which harbors a genetic background predisposed to developing T1D. Mutagenesis in this system is random, but a green fluorescent protein (GFP)-polyA gene trap within the SB transposon enables early detection of mice harboring transposon-disrupted genes. The SB transposon also acts as a molecular tag to, without additional breeding, efficiently identify mutated genes and prioritize mutant mice for further characterization. We show here that the SB transposon is functional in NOD mice and can produce a null allele in a novel candidate gene that increases diabetes incidence. We propose that SB transposon mutagenesis could be used as a complementary strategy to traditional methods to help identify genes that, when disrupted, affect T1D pathogenesis.

Creatine, a naturally occurring nitrogenous organic acid found in animal tissues, has been found to play key roles in the brain including buffering energy supply, improving mitochondrial efficiency, directly acting as an anti-oxidant and acting as a neuroprotectant. Much of the evidence for these roles has been established in vitro or in pre-clinical studies. Here, we examine the roles of creatine and explore the current status of translation of this research into use in humans and the clinic. Some further possibilities for use of creatine in humans are also discussed.

During activity, coordinated vasodilation of microcirculatory networks with upstream supply vessels increases blood flow to skeletal and cardiac muscles and reduces peripheral resistance. Endothelial dysfunction in humans attenuates activity-dependent vasodilation, resulting in exercise-induced hypertension in otherwise normotensive individuals. Underpinning activity-dependent hyperemia is an ascending vasodilation in which the endothelial gap junction protein, connexin (Cx)40, plays an essential role. Because exercise-induced hypertension is proposed as a forerunner to clinical hypertension, we hypothesized that endothelial disruption of Cx40 function in mice may create an animal model of this condition. To this end, we created mice in which a mutant Cx40T152A was expressed alongside wildtype Cx40 selectively in the endothelium. Expression of the Cx40T152A transgene in Xenopus oocytes and mouse coronary endothelial cells in vitro impaired both electric and chemical conductance and acted as a dominant-negative against wildtype Cx40, Cx43, and Cx45, but not Cx37. Endothelial expression of Cx40T152A in Cx40T152ATg mice attenuated ascending vasodilation, without effect on radial coupling through myoendothelial gap junctions. Using radiotelemetry, Cx40T152ATg mice showed an activity-dependent increase in blood pressure, which was significantly greater than in wildtype mice, but significantly less than in chronically hypertensive, Cx40knockout mice. The increase in heart rate with activity was also greater than in wildtype or Cx40knockout mice. We conclude that the endothelial Cx40T152A mutation attenuates activity-dependent vasodilation, producing a model of exercise-induced hypertension. These data highlight the importance of endothelial coupling through Cx40 in regulating blood pressure during activity.

Transporters of the SLC38 family are found in all cell types of the body. They mediate Na+-dependent net uptake and efflux of small neutral amino acids. As a result they are particularly expressed in cells that grow actively, or in cells that carry out si

The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties from the IUPHAR database. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. This compilation of the major pharmacological targets is divided into seven areas of focus: G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors & Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates.

Acetate is a two carbon intermediate in metabolism. It is an accepted marker of astrocytic metabolism, and a substrate for production of metabolites such as glutamine, glutamate and GABA. However, anomalies exist in the current explanations of compartmentation and metabolism of acetate. Here, we investigated these anomalies by examining transport, production and metabolism of acetate. Acetate is a good substrate for the neuronal monocarboxylate transporter MCT2 (KM = 2.58 ± 0.8) and the glial MCT1 but a poor substrate for the glial MCT4. Acetate is accumulated by brain cortical tissue slices to concentrations in excess of those in the media, suggesting active transport, possibly via the sodium dependent SMCT. [2-13C]Acetate is produced from [3-13C]pyruvate, [3-13C]lactate and [1-13C]glucose with the rate of production related to acetyl- CoA levels, which is likely generated in a ubiquitous cytosolic compartment via acetyl-CoA hydrolase. Citrate breakdown occurs in response to demand for acetyl-CoA units; this citrate is not derived from acetate carbon but its fate is influenced by acetate levels. Finally, use of acetate is altered by levels of nicotinamide or NAD+. This suggests that metabolism of acetate is controlled rigorously at the enzyme level, via changes in the acetylation status of acetyl- CoA synthetase and is not regulated by restriction of uptake.

Metabolism of short-chain fatty acids (SCFA) in the brain, particularly that of acetate, appears to occur mainly in astrocytes. The differential use has been attributed to transport, but the extent to which transmembrane movement of SCFA is mediated by transporters has not been investigated systematically. Here we tested the possible contribution of monocarboxylate transporters to SCFA uptake by measuring fluxes with labelled compounds and by following changes of the intracellular pH in Xenopus laevis oocytes expressing the isoforms MCT1, MCT2 or MCT4. All isoforms mediated significant transport of acetate. Formate, however, was transported only by MCT1. The contribution of MCT1 to SCFA transport was determined by using phloretin as a high-affinity inhibitor, which allowed a paired comparison of oocytes with and without active MCT1.

Metabolism in tumour cells is adapted to the demands of a growing cell. The Warburg effect and increased use of glutamine are two well-known adaptations of tumour metabolism. Both require transporters to allow uptake of substrates and efflux of products.

Subcompartments of the plasma membrane are believed to be critical for lymphocyte responses, but few genetic tools are available to test their function. Here we describe a previously unknown X-linked B cell-deficiency syndrome in mice caused by mutations

Amino acids are essential building blocks of all mammalian cells. In addition to their role in protein synthesis, amino acids play an important role as energy fuels, precursors for a variety of metabolites and as signalling molecules. Disorders associated with the malfunction of amino acid transporters reflect the variety of roles that they fulfil in human physiology. Mutations of brain amino acid transporters affect neuronal excitability. Mutations of renal and intestinal amino acid transporters affect whole-body homoeostasis, resulting in malabsorption and renal problems. Amino acid transporters that are integral parts of metabolic pathways reduce the function of these pathways. Finally, amino acid uptake is essential for cell growth, thereby explaining their role in tumour progression. The present review summarizes the involvement of amino acid transporters in these roles as illustrated by diseases resulting from transporter malfunction.

The neutral amino acid transporter B°-like from rat kidney, previously reconstituted in liposomes, was identified as B°AT1 by a specific antibody. Collectrin was present in the brush-border extract but not in functionally active proteoliposomes, indicat

Xenopus oocytes are a versatile expression system particularly suited for membrane transporters and channels. Oocytes have little background activity and therefore offer a very high signal-to-noise ratio for transporter and channel characterization. This chapter provides an overview of the basic methods used for the analysis of membrane transporters in this system, including preparation of oocytes, assays of transport activity, protocols for immunostaining and fluorescence microscopy, and other assays to study surface expression.

The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na� and Cl�-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na�-binding sites and a putative Cl�-binding site. Mutation of all residues in the two proposed Na�-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na�-binding site 1) and mutants S20A, S20G, S20G/G405S (Na�-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na�-binding site 1 caused a decrease of the Na�-K0.5, whereas mutations of site 2 increased the K0.5. Mutation of Na�-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates 36Cl� demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na�/1Cl�- proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria.

The emergence and spread of chloroquine-resistant Plasmodium falciparum malaria parasites has been a disaster for world health. Resistance is conferred by mutations in the Chloroquine Resistance Transporter (PfCRT), an integral membrane protein localized

A novel inhibitor of lactate transport, AR-C122982, was used to study the effect of inhibiting the monocarboxylate transporters MCT1 and MCT2 on cortical brain slice metabolism. We studied metabolism of l-[3-13C]lactate, and d-[1-13C]glucose under a range of conditions. Experiments using l-[3-13C]lactate showed that the inhibitor AR-C122982 altered exchange of lactate. Under depolarizing conditions, net flux of label from d-[1-13C]glucose was barely altered by 10 or 100 nM AR-C122982. In the presence of AMPA or glutamate there were increases in net flux of label and metabolic pool sizes. These data suggest lactate may supply compartments in the brain not usually directly accessed by glucose. In general, it would appear that movement of lactate between cell types is not essential for metabolic activity, with the heavy metabolic workloads imposed being unaffected by inhibition of MCT1 and MCT2. Further experiments investigating the mechanism of operation of AR-C122982 are necessary to corroborate this finding.