Plasma membrane transporters Alp1 and Nrt1 mediate the uptake of benzamidine conjugates in fungi.

ABSTRACTMembrane transport proteins perform crucial roles in cell physiology. The obligate intracellular parasite Plasmodium falciparum, an agent of human malaria, relies on membrane transport proteins for the uptake of nutrients from the host, disposal of metabolic waste, exchange of metabolites between organelles, and generation and maintenance of transmembrane electrochemical gradients for its growth and replication within human erythrocytes. Despite their importance for Plasmodium cellular physiology, the functional roles of a number of membrane transport proteins remain unclear, which is particularly true for orphan membrane transporters that have no or limited sequence homology to transporter proteins in other evolutionary lineages. Therefore, in the current study, we applied endogenous tagging, targeted gene disruption, conditional knockdown, and knockout approaches to investigate the subcellular localization and essentiality of six membrane transporters during intraerythrocytic development of P. falciparum parasites. They are localized at different subcellular structures—the food vacuole, the apicoplast, and the parasite plasma membrane—and four out of the six membrane transporters are essential during asexual development. Additionally, the plasma membrane resident transporter 1 (PMRT1; PF3D7_1135300), a unique Plasmodium-specific plasma membrane transporter, was shown to be essential for gametocytogenesis and functionally conserved within the genus Plasmodium. Overall, we reveal the importance of four orphan transporters to blood stage P. falciparum development, which have diverse intracellular localizations and putative functions.

The equilibrative nucleoside transporters, hENT1 and CeENT1 from humans and Caenorhabditis elegans, respectively, are inhibited by nanomolar concentrations of dipyridamole and share a common 11-transmembrane helix (TM) topology. Random mutagenesis and screening by functional complementation in yeast for clones with reduced sensitivities to dipyridamole yielded mutations at Ile429 in TM 11 of CeENT1 and Met33 in TM 1 of hENT1. Mutational analysis of the corresponding residues of both proteins suggested important roles for these residues in competitive inhibition of hENT1 and CeENT1 by dipyridamole. To verify the roles of these residues in dipyridamole interactions, hENT2, which naturally exhibits low dipyridamole sensitivity, was mutated to contain side chains favorable for high affinity dipyridamole binding (i.e. a Met at the TM 1 and/or an Ile at the TM 11 positions). The single mutants exhibited increased hENT2 sensitivity to inhibition by dipyridamole, and the double mutant was the most sensitive, with an IC50 value that was only 2% of that of wild type. Functional analysis of the TM 1 and 11 mutants of hENT1 and CeENT1 revealed that Ala and Thr in the TM 1 and 11 positions, respectively, impaired uridine and adenosine transport and that Leu442 of hENT1 was involved in permeant selectivity. Mechanistic and structural models of dipyridamole interactions with the TM 1 and 11 residues are proposed. This study demonstrated that the corresponding residues in TMs 1 and 11 of hENT1, hENT2, and CeENT1 are important for dipyridamole interactions and nucleoside transport.

![Figure][1] J. Patrick Kampf Torrey Pines Institute for Molecular Studies, San Diego, California ![Figure][1] Alan M. Kleinfeld Torrey Pines Institute for Molecular Studies, San Diego, California akleinfeld{at}tpims.org The mechanism of free fatty acid (FFA)

The central nervous system (CNS) is segregated from the circulating blood and peripheral tissues by endothelial and epithelial barriers. To overcome refractory CNS diseases, it is important to understand the membrane transport systems of drugs and the endogenous compounds that relate to the pathogenesis of CNS diseases at these barriers. The endothelial barrier in the brain is the blood-brain barrier (BBB). Our studies clarified the efflux transport of prostaglandin E2 (PGE2), a modulator of neural excitation and inflammatory responses, across the BBB via plasma membrane transporters such as organic anion transporter 3 (Oat3) and multidrug resistance-associated protein 4 (Mrp4). This efflux transport was attenuated by peripheral inflammation or cerebral treatment with neuroexcitatory l-glutamate, suggesting that BBB-mediated PGE2 elimination was altered under several pathological conditions. We also examined excitatory amino acid transporter (EAAT) 1 and 3 as l-glutamate efflux transporters of the inner blood-retinal barrier (BRB) and blood-cerebrospinal barrier. It was considered that these efflux membrane transporters participated in the homeostasis of neuroexcitatory and neuroinflammatory responses in the brain and retina. Moreover, we identified connexin 43 (Cx43) hemichannels as a new membrane transport system that is activated under pathological conditions and recognizes several monocarboxylate drugs, such as valproate. As it is expected that the action of these membrane transporters across the CNS barriers is of great importance in understanding the pathology of various neuroexcitatory diseases, our studies should contribute to the establishment of therapeutic strategies for refractory CNS diseases.

Clinical Pharmacology & Therapeutics (2010) 87 1, 3–7. doi:10.1038/clpt.2009.239 “False facts are highly injurious to the progress of science, for they often endure long; but false views, if supported by some evidence, do little harm, for everyone takes a salutary pleasure in proving their falseness.” —Charles Darwin, The Descent of Man (1872) Membrane-localized transport proteins have evolved to catalyze migration or expulsion of an endogenous or exogenous molecule across biological membranes. Such evolution has been necessary because membrane lipid bilayers would otherwise present virtually impenetrable barriers to most (hydrophilic) molecules or, alternatively, permit excessive intracellular accumulation. Hence, it became possible to regulate the composition of intracellular and extracellular fluids with the advent of membrane transport proteins (“transporters”). Moreover, such regulation was made progressively more sophisticated as more types of proteins evolved to transport a wide array of molecular species.1 Contemporary bioinformatics analyses have provided an emerging picture of how the majority of membrane transporters probably evolved, and such studies have allowed postulation of the specific pathways taken for their appearance.2 The ancestral precursors of most membrane transporters were simple peptide channels, and the pathways most frequently taken were evidently tandem intragenic duplications that gave rise to larger helical bundles with the potential to form discrete stereospecific intramembrane substrate binding sites. They could also be constrained for coupling to other transport processes and, through conformational coupling, were subject to control by a superimposed primary energy-yielding process such as adenosine triphosphate (ATP) hydrolysis. Until recently, the number of identified membrane transporters in mammalian species was relatively modest, but in the postgenomic era this number is now rapidly increasing; based on homology/motif searching and semiautomated curation, the TransportDB website currently lists as many as 1,022 transporters for Homo sapiens alone (http://www.membranetransport.org). Although most of these transporters have unknown physiological function, several have gained interest within the scientific community at an ever-increasing rate, partly because it is recognized that drugs may “hitchhike” on these proteins that normally act on endogenous substrates. Although the concept of a transporter-mediated movement of certain drugs into and out of cells had already been proposed by the 1950s,3 it was not until the human membrane transporter ABCB1 (also known as P-glycoprotein, MDR1) was cloned in 1987 that the drug transporter field truly took off.4 Since then, the physiological and pharmacological roles of several of these proteins have been studied in a wide range of therapeutic areas, including oncology, cardiovascular diseases, infectious diseases, and diabetes. The primary focus of many initial studies was the evaluation of the impact of membrane transporters on the development of cellular resistance to drugs and on their role in the absorption and disposition of xenobiotics. The vast majority of transporter research has been conducted on the ATP-binding cassette (ABC) transporters, a major superfamily of membrane transporters that consists of at least 48 proteins that bind and hydrolyze ATP so as to drive the efflux of various compounds out of the cell. Although the role of ABC transporters in drug resistance has historically been a major focus of research, recent years have seen a greater emphasis on studies devoted to the so-called solute carriers, which comprise more than 300 proteins organized into 47 distinct families and which are involved in the uptake of molecules into cells. These transporters do not require ATP to function and are commonly classified as passive transporters, ion-coupled transporters, or exchangers. One contributor to the apparent historical lack of interest in solute carrier research is the long-held belief that most xenobiotics are transported across membranes via passive diffusion at a rate related to their hydrophobicity.5 Not only is this accepted paradigm itself poorly supported by experimental data, but evidence accumulated over the past decade now supports the notion that solute carrier-mediated migration of drugs across the highly organized lipid bilayers of biological membranes is the predominant mechanism of cellular uptake, even for hydrophobic agents.1 The two most commonly studied solute carrier families are the SLCO family of genes that encode organic anion-transporting polypeptides (OATPs) and the SLC22A family of genes that encode organic cation transporters (OCTs) and organic anion transporters (OATs). The first solute carrier to be cloned was the rodent Oatp1a1 transporter,6 with the rodent Oct1 and Oat1 cloned shortly thereafter.7,8,9 Whereas the early in vitro and in vivo studies were capable of functionally characterizing various solute carriers, it was the actual cloning of these proteins in the early 1990s that really allowed for the massive surge in understanding and exploiting them in a therapeutic context. In this issue of Clinical Pharmacology & Therapeutics, Hagenbuch provides a historical perspective on the early studies on hepatic and renal solute carriers and the subsequent boom in studies evaluating their role in regulating the pharmacokinetics and pharmacodynamics of drugs.10 One line of evidence supporting the importance of solute carrier-mediated cellular uptake of drugs is that there are now many cases in which drugs are known to be taken up into cells via defined transporters expressed in specific tissues or organs. An example of this was the identification of fexofenadine as a substrate for OATP1A2, a solute carrier that is localized in enterocytes and may account for the absorption of fexofenadine after oral administration.11 Another line of reasoning demonstrating that drugs can be transported across membranes into cells by solute carriers is the notion that many drugs specifically accumulate in particular tissues, that their degree of accumulation is often greater than any possible number of intracellular binding sites, and that this process can be affected by chemical inhibitors of solute carriers. For example, a recent study reported in this journal indicated that the ability of the anticancer drug cisplatin to specifically accumulate in renal proximal tubular cells and subsequently cause severe, dose-limiting nephrotoxicity is determined by a tissue-specific overexpression in the kidney of certain OCTs.12 This type of information has supported the idea of possibly exploiting tissue-specific transporter distribution for preventing drug-induced toxicity. Indeed, concomitant use of a known inhibitor of renal OATs, probenecid, with the antiviral agent cidofovir has now been recommended in order to decrease the incidence and severity of cidofovir-induced nephrotoxicity.13,14 Likewise, as discussed by Niemi,15 a wealth of knowledge has accumulated within the past few years pointing to a critical role of several polymorphic transporters in the disposition, toxicity, and activity of statins (or HMG-CoA reductase inhibitors), a class of drugs that lower cholesterol levels in people with or at risk of cardiovascular disease. There are sometimes complications in translating preclinical findings to the clinic, and these can be explained in part by differences in tissue localization and/or substrate recognition of transporters between humans and animals, although future studies could use humanized mice to possibly improve animal-to-human translational findings.10 The use of animal models deficient in certain transporters has historically provided invaluable information on the normal physiological function of these proteins. For example, mice deficient in ABCB1, a transporter involved in the extrusion of structurally diverse toxic substances from cells, are phenotypically normal but are extremely sensitive to certain neurotoxins.16 These studies led to the characterization of an important role of ABCB1 in the transport of numerous prescription drugs across the blood-brain barrier. In this issue of the Journal, the Point/Counterpoint pieces by Marchi et al. and Cascorbi, respectively, provide an example of how this transporter, which was assumed to be an important target responsible for drug-refractory epilepsy on the basis of preclinical studies, may not be as critical in humans as formerly held.17,18 Investigations into how ABC transporters and solute carriers impact drug distribution, toxicity, efficacy, and drug resistance have become the focus of many efforts to now rapidly expand knowledge in this field. The increasing awareness of the importance of these transporters is emphasized by the recent development of the International Transporter Consortium—described in this issue by Huang et al.—which is made up of scientists from academia, industry, and government who have an interest in studying and developing new tools for understanding the role of transporters in drug discovery, development, and regulatory approval.19 Moreover, a summary of the accomplishments of the National Institutes of Health–funded Pharmacogenomics of Membrane Transporters (PMT) project, described by Kroetz et al., provides ample evidence of how public funding in transporter research can improve multidisciplinary and translational research and benefit a wide variety of therapeutic areas.20 The PMT project has significantly contributed to the discovery of inherited variability in transporter genes, including single-nucleotide polymorphisms, many of which can lead to changes in protein expression and function and affect the pharmacokinetic and pharmacodynamic profiles of substrate drugs. Current translational research has also begun to utilize transporters as a way of allowing formerly unavailable administration routes of substrate drugs. For example, the expression of several ABC transporters on the apical surface of epithelial cells of the gastrointestinal tract is known to influence intestinal drug absorption and limit oral bioavailability of many drugs, including several important anticancer agents such as the taxanes docetaxel and paclitaxel, that could historically be administered only by intravenous infusion. As described by Koolen et al., the usage of chemical inhibitors of these transporters has provided a means for administering taxanes orally in the clinic, which is a more practical and convenient method that will allow treatment of patients in the future on an outpatient basis.21 Another important area of current transporter research is evolving around the discovery and development of agents that directly target specific transporters. Last year, two publications in CPT highlighted the utility of dapagliflozin, an inhibitor of the glucose transporter SGLT2 (SLC5A2), as a non-insulin based therapeutic strategy to reduce the re-uptake of glucose from the urine in patients with type 2 diabetes.22,23 In addition, the potential use of inhibitors for the Niemann–Pick C1-like 1 (NPC1L1) transporter, such as ezetimibe, in treating hypercholesterolemia by preventing the intestinal absorption of cholesterol from diet presents an interesting approach to treating this condition, as discussed by Betters and Yu.24 These authors propose a three-pronged drug approach in the treatment of hypercholesterolemia, including inhibition of intestinal absorption (with ezetimibe), inhibition of biosynthesis (with statins), and increased excretion of cholesterol (with an activator of ABC transporters). Finally, in the field of parasitic protozoa treatment, the concept of parasite-specific transporters as targets for drug delivery or drug targeting offers some very exciting opportunities, as highlighted by Landfear.24,25 Drugs can be designed to be specific for parasitic transporters, thus limiting or reducing toxicities in humans. Furthermore, drugs can be developed to inhibit parasitic transporters that are necessary for the delivery of nutrients to the parasite. Although more research must be conducted to confirm a critical role of transporters in drug effects and determine how to utilize transporters to improve treatment, reduce interindividual pharmacokinetic variability, and minimize toxicity, the interest and involvement of all areas of research (academy, industry, and government) give hope that this field will lead to even greater beneficial findings. The research conducted in the past several decades has led to a greater understanding of what happens to a drug once it enters the body, as well as the mechanisms underlying drug transport. Given the sheer magnitude of the number of transporters in humans identified thus far, it is not difficult to imagine that the work done so far represents, at best, the tip of the iceberg. Given the role of membrane transporters for drugs, future studies that can clarify discrepancies and further identify possible targets for therapeutics in humans have the potential to advance the quality of life and improve treatment outcomes of a wide variety of diseases. The authors declared no conflict of interest.

Light is a vital regulator that controls physiological and cellular responses to regulate plant growth, development, yield, and quality. Light is the driving force for electron and ion transport in the thylakoid membrane and other membranes of plant cells. In different plant species and cell types, light activates photoreceptors, thereby modulating plasma membrane transport. Plants maximize their growth and photosynthesis by facilitating the coordinated regulation of ion channels, pumps, and co-transporters across membranes to fine-tune nutrient uptake. The signal-transducing functions associated with membrane transporters, pumps, and channels impart a complex array of mechanisms to regulate plant responses to light. The identification of light responsive membrane transport components and understanding of their potential interaction with photoreceptors will elucidate how light-activated signaling pathways optimize plant growth, production, and nutrition to the prevailing environmental changes. This review summarizes the mechanisms underlying the physiological and molecular regulations of light-induced membrane transport and their potential interaction with photoreceptors in a plant evolutionary and nutrition context. It will shed new light on plant ecological conservation as well as agricultural production and crop quality, bringing potential nutrition and health benefits to humans and animals.

Although its immunomodulatory properties make thymopentin (TP5) appealing, its rapid metabolism and inactivation in the digestive system pose significant challenges for global scientists. PEGylated niosomal nanocarriers are hypothesized to improve the physicochemical stability of TP5, and to enhance its intestinal permeability for oral administration. TP5-loaded PEGylated niosomes were fabricated using the thin film hydration method. Co-cultured Caco-2 and HT29 cells with different ratios were screened as in vitro intestinal models. The cytotoxicity of TP5 and its formulations were evaluated using an MTT assay. The cellular uptake and transport studies were investigated in the absence or presence of variable inhibitors or enhancers, and their mechanisms were explored. All TP5 solutions and their niosomal formulations were nontoxic to Caco-2 and HT-29 cells. The uptake of TP5-PEG-niosomes by cells relied on active endocytosis, exhibiting dependence on time, energy, and concentration, which has the potential to significantly enhance its cellular uptake compared to TP5 in solution. Nevertheless, cellular transport rates were similar between TP5 in solution and its niosomal groups. The cellular transport of TP5 in solution was carried out mainly through MRP5 endocytosis and a passive pathway and effluxed by MRP5 transporters, while that of TP5-niosomes and TP5-PEG-niosomes was carried out through adsorptive- and clathrin-mediated endocytosis requiring energy. The permeability and transport rate was further enhanced when EDTA and sodium taurocholate were used as the penetration enhancers. This research has illustrated that PEG-niosomes were able to enhance the cellular uptake and maintain the cellular transport of TP5. This study also shows this formulation's potential to serve as an effective carrier for improving the oral delivery of peptides.

The treatment of common psychiatric disorders like major depression, schizophrenia and bipolar disorder is characterized by low efficacy and variability in the case of depression and bipolar disorder, and of undesirable side effects in the case of schizophrenia. One of the explanations is that the drug may not be reaching its site of action, at concentrations that are high enough to provoke a response. On the other hand, poor elimination of the drug from the body may lead to high plasma concentrations, which may cause undesirable side effects. Variations in membrane transport at the blood-brain barrier might affect the concentra- tion of psychotropic drugs at their site of action. In organs such as the liver and kidney, variations in membrane transport may affect drug elimination. Using a parallel artificial membrane assay, 31 commonly used psychotropic drugs were screened for their ability to penetrate cell membranes by passive diffusion. Using custom made TaqMan® low-density gene expression arrays, the mRNA expression of 90 drug transporters was analyzed in organs relevant for drug pharmacokinetics and in human primary brain cells. HEK293 cells overexpressing organic cation transporters were used to study the transporter-mediated cellular uptake of psychotropic drugs. Finally, the immortalized human brain microvascular endothelial cell line, hCMEC/D3, was used as a blood-brain barrier model to study influx transport. In human primary brain microvascular endothelial cells (HBMECs), the expression of organic cation transporters was substantially lower than in other organs like the liver and the kidney. Nonetheless organic cation transporters were detected in HBMECs. OCTN2 was the organic cation transporter with the highest expression, followed by OCTN1, OCT1 and OCT3. Amisulpride, sulpiride, sultopride and tiapride were identified as drugs with low mem- brane permeability, which may require influx transport to reach their site of action in the brain. Amisulpride and sulpiride were identified in vitro as substrates of the organic caiton trasnporters of the SLC22 family and may depend on organic cation mediated transport to cross the blood-brain barrier. The presence of a carrier-mediated trans- port mechanism for the uptake of amisulpride and sulpiride was confirmed in the brain endothelial cell line model hCMEC/D3. Furthermore, absorption and elimination of amisulpride and sulpiride may also depend on organic cation transporters. OCT1 may contribute to the billiary elimination of amisulpride and sulpiride. In addition, the transporters OCT2, MATE1 and MATE2-K may contribute for the renal elimination of amisulpride and sulpiride in the proximal tubule epithelium. Common genetic polymorphisms on the OCT1 gene were found to affect the cellular uptake of amisulpride and sulpiride. The majority of the psychotropic drugs, like amitriptyline, have high membrane perme- ability and may not benefit from drug transporters to permeate cellular barriers in the in vitro models used in this work. However, these drugs can still interact strongly with membrane transporters, like OCT1. Clinical studies, providing in vivo evidence for the interaction of high permeability drugs with membrane transporters, will be needed in the future. Weak basic psychotropic drugs may inhibit the OCT1-mediated uptake of other important drugs, like morphine. The psychotropic drugs amitriptyline, clomipramine, imipramine and fluoxetine, and also irinotecan, ondansetron and verapamil, inhibited the OCT1-mediated uptake of morphine at therapeutically relevant concentrations. Furthermore, the effect of genetic polymorphisms in the OCT1 gene on the OCT1- mediated uptake of the biogenic amine tyramine was studied. In addition, an MDCK II cell line carrying a site for targeted chromosomal gene integration was developed. This model should in the future enable the analysis of the effects of genetic polymorphisms on drug transport by efflux transporters, which are present at the blood-brain barrier. In conclusion, this study demonstrates that influx transporters may mediate the uptake of psychotropic drugs with low membrane permeability like amisulpride and sulpiride, and may influence their pharmacokinetics and distribution to the brain. This work, and the tools which were developed here, can serve as a basis for further work on the role of organic cation transporters at the blood-brain barrier, and to study in more detail the role of organic cation transporters in the pharmacokinetics of amisulpride and sulpiride.

BACKGROUNDCardiac and hepatic functionality are intertwined in a multifaceted relationship. Pathologic processes involving one may affect the other through a variety of mechanisms, including hemodynamic and membrane transport effects.AIMTo better understand the effect of extrahepatic cholestasis on regulations of membrane transporters involving digoxin and its implication for digoxin clearance.METHODSTwelve adult rats were included in this study; baseline hepatic and renal laboratory values and digoxin pharmacokinetic (PK) studies were established before evenly dividing them into two groups to undergo bile duct ligation (BDL) or a sham procedure. After 7 d repeat digoxin PK studies were completed and tissue samples were taken to determine the expressions of cell membrane transport proteins by quantitative western blot and real-time polymerase chain reaction. Data were analyzed using SigmaStat 3.5. Means between pre-surgery and post-surgery in the same experimental group were compared by paired t-test, while independent t-test was employed to compare the means between sham and BDL groups.RESULTSDigoxin clearance was decreased and liver function, but not renal function, was impaired in BDL rats. BDL resulted in significant up-regulation of multidrug resistance 1 expression in the liver and kidney and its down-regulation in the small intestine. Organic anion transporting polypeptides (OATP)1A4 was up-regulated in the liver but down-regulated in intestine after BDL. OATP4C1 expression was markedly increased in the kidney following BDL.CONCLUSIONThe results suggest that cell membrane transporters of digoxin are regulated during extrahepatic cholestasis. These regulations are favorable for increasing digoxin excretion in the kidney and decreasing its absorption from the intestine to compensate for reduced digoxin clearance due to cholestasis.

The decline in activity of distinct membrane transport systems was followed during in vitro maturation of sheep reticulocytes, namely the sodium pump (measured as specific ouabain binding sites), Na+-glycine cotransport, and the nucleoside transporter (measured as specific nitrobenzylthioinosine binding sites). Certain features of this maturation-associated decline in membrane transport are clarified. Thus, the apparent retardation of loss by metabolic (ATP) depletion, reported previously for the sodium pump and Na+-glycine cotransport, is applicable also to the decline in nucleoside transport. The absolute losses, as well as relative effects of ATP depletion, are different for the three distinct systems. Inhibitors of membrane recycling and (or) intracellular processing, such as chloroquine, as well as ATP depletion, prevent not only the loss but also cause a transient increase in nucleoside transport sites apparent at the surface. Proteolytic processing, at least in the case of the nucleoside transporter, is probably also involved since leupeptin retards the loss in binding sites. Protection against the decline in transporters can also be affected by specific ligands as evidenced in ouabain protection of sodium pump sites. The results provide evidence that membrane transporter recycling is a fundamental process underlying the energy-dependent, maturation-associated loss in membrane transport functions.

Erythrocytes are endowed with functional entities that support either cellular functions or the systemic delivery of O2 from lung to tissue and removal of CO2 from tissue to lung. The latter depend largely on the blood's circulatory capacity. They are associated, respectively, with cytosolic haemoglobin and the major membrane polypeptide band 3 (anion exchanger 1, AE1). As a membrane transporter, AE1 mediates Cl-/HCO3- exchange, thus enhancing the blood capacity for carrying CO2 and for acid-base homeostasis. By interacting with lipids and proteins, the multifunctional AE1 tethers the membrane cytoskeleton multiprotein complex to the membrane and confers upon erythrocytes both mechanical and viscoelastic properties. Those in turn allow cells to withstand the shear forces of circulation and squeeze through capillaries. Most other major membrane transporters are apparently essential for maintaining a stable erythrocyte cell shape and flexibility via a functional membrane cytoskeleton. These include the membrane transporters of glucose, nucleoside and purine for fueling the Na/K and Ca pumps via ATP production, and of amino acid and oxidized glutathione transport for maintaining the cell redox status. All membrane transporters detected in mature erythrocytes are synthesized early in erythrocyte differentiation. Their contribution to erythrocyte and to systemic physiology is presently being re-assessed by targeted gene disruption and replacement. For example, organisms with reduced or disrupted AE1 gene expression showed major erythrocyte instabilities and defective anion exchange capacity and acidosis, but remain alive.

Background: Bilirubin is a highly-hydrophobic tetrapyrrole which binds to plasma albumin. It is conjugated in the liver to glucuronic acid, and the water-soluble glucuronides are excreted in urine and bile. The membrane transporters of bilirubin diglucuronide are well-known. Still undefined are however the transporters performing the uptake of bilirubin from the blood into the liver, a process known to be fast and not rate-limited. The biological importance of this process may be appraised by considering that in normal adults 200–300 mg of bilirubin are produced daily, as a result of the physiologic turnover of hemoglobin and cellular cytochromes. Nevertheless, research in this field has yielded controversial and contradicting results. We have undertaken a systematic review of the literature, believing in its utility to improve the existing knowledge and promote further advancements.Methods: We have sourced the PubMed database until 30 June 2017 by applying 5 sequential searches. Screening and eligibility criteria were applied to retain research articles reporting results obtained by using bilirubin molecules in membrane transport assays in vitro or by assessing serum bilirubin levels in in vivo experiments.Results: We have identified 311 articles, retaining 44, reporting data on experimental models having 6 incremental increases of complexity (isolated proteins, membrane vesicles, cells, organ fragments, in vivo rodents, and human studies), demonstrating the function of 19 membrane transporters, encoded by either SLCO or ABC genes. Three other bilirubin transporters have no gene, though one, i.e., bilitranslocase, is annotated in the Transporter Classification Database.Conclusions: This is the first review that has systematically examined the membrane transporters for bilirubin and its conjugates. Paradoxically, the remarkable advancements in the field of membrane transport of bilirubin have pointed to the elusive mechanism(s) enabling bilirubin to diffuse into the liver as if no cellular boundary existed.

Membrane transporters carry key metabolites across the cell membrane and, from a resource standpoint, are hypothesized to be produced when necessary. The expression of membrane transporters in metabolic pathways is often upregulated by the transporter substrate. In E. coli, such systems include for example the lacY, araFGH, and xylFGH genes, which encode for lactose, arabinose, and xylose transporters, respectively. As a case study of a minimal system, we build a generalizable physical model of the xapABR genetic circuit, which features a regulatory feedback loop via membrane transport (positive feedback) and enzymatic degradation (negative feedback) of an inducer. Dynamical systems analysis and stochastic simulations show that the membrane transport makes the model system bistable in certain parameter regimes. Thus, it serves as a genetic “on-off” switch, enabling the cell to only produce a set of metabolic enzymes when the corresponding metabolite is present in large amounts. We find that the negative feedback from the degradation enzyme does not significantly disturb the positive feedback from the membrane transporter. We investigate hysteresis in the switching and discuss the role of cooperativity and multiple binding sites in the model circuit. Fundamentally, this work explores how a stable genetic switch for a set of enzymes is obtained from transcriptional auto-activation of a membrane transporter through its substrate.

Membrane transporters carry key metabolites across the cell membrane and, from a resource standpoint, are hypothesized to be produced when necessary. The expression of membrane transporters in metabolic pathways is often upregulated by the transporter substrate. In E. coli, such systems include for example the lacY, araFGH, and xylFGH genes, which encode for lactose, arabinose, and xylose transporters, respectively. As a case study of a minimal system, we build a generalizable physical model of the xapABR genetic circuit, which features a regulatory feedback loop via membrane transport (positive feedback) and enzymatic degradation (negative feedback) of an inducer. Dynamical systems analysis and stochastic simulations show that the membrane transport makes the model system bistable in certain parameter regimes. Thus, it serves as a genetic "on-off" switch, enabling the cell to only produce a set of metabolic enzymes when the corresponding metabolite is present in large amounts. We find that the negative feedback from the degradation enzyme does not significantly disturb the positive feedback from the membrane transporter. We investigate hysteresis in the switching and discuss the role of cooperativity and multiple binding sites in the model circuit. Fundamentally, this work explores how a stable genetic switch for a set of enzymes is obtained from transcriptional auto-activation of a membrane transporter through its substrate.

Membrane transporters are a large group of proteins that span cell membranes and contribute to critical cell processes, including delivery of essential nutrients, ejection of waste products, and assisting the cell in sensing environmental conditions. Obtaining an accurate and specific annotation of the transporter proteins encoded by a micro-organism can provide details of its likely nutritional preferences and environmental niche(s), and identify novel transporters that could be utilized in small molecule production in industrial biotechnology. The Transporter Automated Annotation Pipeline (TransAAP) (http://www.membranetransport.org/transportDB2/TransAAP_login.html) is a fully automated web service for the prediction and annotation of membrane transport proteins in an organism from its genome sequence, by using comparisons with both curated databases such as the TCDB (Transporter Classification Database) and TDB, as well as selected Pfams and TIGRFAMs of transporter families and other methodologies. TransAAP was used to annotate transporter genes in the prokaryotic genomes in the National Center for Biotechnology Information (NCBI) RefSeq; these are presented in the transporter database TransportDB (http://www.membranetransport.org) website, which has a suite of data visualization and analysis tools. Creation and maintenance of a bioinformatic database specific for transporters in all genomic datasets is essential for microbiology research groups and the general research/biotechnology community to obtain a detailed picture of membrane transporter systems in various environments, as well as comprehensive information on specific membrane transport proteins.