Functional reconstitution of SdcS, a Na+-coupled dicarboxylate carrier protein from Staphylococcus aureus

In Staphylococcus aureus, the transport of dicarboxylates is mediated in part by the Na+-linked carrier protein SdcS. This transporter is a member of the divalent-anion/Na+ symporter (DASS) family, a group that includes the mammalian Na+/dicarboxylate cotransporters NaDC1 and NaDC3. In earlier work, we cloned and expressed SdcS in Escherichia coli and found it to have transport properties similar to those of its eukaryotic counterparts (J. A. Hall and A. M. Pajor, J. Bacteriol. 187:5189-5194, 2005). Here, we report the partial purification and subsequent reconstitution of functional SdcS into liposomes. These proteoliposomes exhibited succinate counterflow activity, as well as Na+ electrochemical-gradient-driven transport. Examination of substrate specificity indicated that the minimal requirement necessary for transport was a four-carbon terminal dicarboxylate backbone and that productive substrate-transporter interaction was sensitive to substitutions at the substrate C-2 and C-3 positions. Further analysis established that SdcS facilitates an electroneutral symport reaction having a 2:1 cation/dicarboxylate ratio. This study represents the first characterization of a reconstituted Na+-coupled DASS family member, thus providing an effective method to evaluate functional, as well as structural, aspects of DASS transporters in a system free of the complexities and constraints associated with native membrane environments.     

Functional characterization of a Na+-coupled dicarboxylate transporter from Bacillus licheniformis

The Na⁺-coupled dicarboxylate transporter, SdcL, from Bacillus licheniformis is a member of the divalent anion/Na⁺ symporter (DASS) family that includes the bacterial Na⁺/dicarboxylate cotransporter SdcS (from Staphyloccocus aureus) and the mammalian Na⁺/dicarboxylate cotransporters, NaDC1 and NaDC3. The transport properties of SdcL produced in Escherichia coli are similar to those of its prokaryotic and eukaryotic counterparts, involving the Na⁺-dependent transport of dicarboxylates such as succinate or malate across the cytoplasmic membrane with a K_m of ～ 6 μM. SdcL may also transport aspartate, α-ketoglutarate and oxaloacetate with low affinity. The cotransport of Na⁺ and dicarboxylate by SdcL has an apparent stoichiometry of 2:1, and a K_0.5 for Na⁺ of 0.9 mM. Our findings represent the characterization of another prokaryotic protein of the DASS family with transport properties similar to its eukaryotic counterparts, but with a broader substrate specificity than other prokaryotic DASS family members. The broader range of substrates carried by SdcL may provide insight into domains of the protein that allow a more flexible or larger substrate binding pocket.     

Protein kinase C-mediated regulation of the renal Na(+)/dicarboxylate cotransporter, NaDC-1.

The Na(+)/dicarboxylate cotransporter of the renal proximal tubule, NaDC-1, reabsorbs Krebs cycle intermediates, such as succinate and citrate, from the tubular filtrate. Although long-term regulation of this transporter by chronic metabolic acidosis and K(+) deficiency is well documented, there is no information on acute regulation of NaDC-1. In the present study, the transport of succinate in Xenopus oocytes expressing NaDC-1 was inhibited up to 95% by two activators of protein kinase C, phorbol 12-myristate, 13-acetate (PMA) and sn-1, 2-dioctanoylglycerol (DOG). Activation of protein kinase A had no effect on NaDC-1 activity. The inhibition of NaDC-1 transport by PMA was dose-dependent, and could be prevented by incubation of the oocytes with staurosporine. Mutations of the two consensus protein kinase C phosphorylation sites in NaDC-1 did not affect inhibition by PMA. The inhibitory effects of PMA were partially prevented by cytochalasin D, which disrupts microfilaments and endocytosis. PMA treatment was also associated with a decrease of approximately 30% in the amount of NaDC-1 protein found on the plasma membrane. We conclude that the inhibition of NaDC-1 transport activity by PMA occurs by a combination of endocytosis and inhibition of transport activity.     

Stimulation of renal Na+ dicarboxylate cotransporter 1 by Na+/H+ exchanger regulating factor 2, serum and glucocorticoid inducible kinase isoforms, and protein kinase B

Renal tubular citrate transport is accomplished by electrogenic Na(+) coupled dicarboxylate transporter NaDC-1, a carrier subjected to regulation by acidosis. Trafficking of the Na(+)/H(+) exchanger NHE3 is controlled by NHE regulating factors NHERF-1 and NHERF-2 and the serum and glucocorticoid inducible kinase SGK1. To test for a possible involvement in NaDC-1 regulation, mRNA encoding NaDC-1 was injected into Xenopus oocytes with or without cRNA encoding NHERF-1, NHERF-2, SGK1, SGK2, SGK3, and/or the constitutively active form of the related protein kinase B ((T308,S473D)PKB). Succinate induced inward currents (I(succ)) were taken as a measure of transport rate. Coexpression of neither NHERF-1 nor NHERF-2 in NaDC-1 expressing oocytes significantly altered I(succ). On the other hand, coexpression of SGK1, SGK3, and (T308,S473D)PKB stimulated I(succ), an effect further stimulated by additional coexpression of NHERF-2 but not of NHERF-1. The action of the kinases and NHERF-2 may link urinary citrate excretion to proximal tubular H(+) secretion.     

Interactions of benzylpenicillin and non-steroidal anti-inflammatory drugs with the sodium-dependent dicarboxylate transporter NaDC-3.

Sodium-dependent dicarboxylate transporters located in the basolateral membrane (NaDC-3) of renal proximal tubule cells maintain the driving force for exchange of organic anions and drugs against alpha-ketoglutarate via organic anion transporters OAT1 and OAT3. So far, information on direct interaction of drugs with the cloned NaDC-3 was missing. Here we tested the interaction of non-steroidal anti-inflammatory drugs (NSAIDs) and benzylpenicillin with NaDC-3 cloned from winter flounder (fNaDC-3) and human (hNaDC-3) kidneys. Flufenamate and benzylpenicillin inhibited [14C]succinate uptake in oocytes expressing fNaDC-3. Flufenamate elicited Na(+)-dependent currents in oocytes expressing fNaDC-3 with a reversal potential around -60 mV. Raising extracellular K+ concentration depolarized fNaDC3-expressing oocytes more in the presence of flufenamate than in its absence, an effect not seen with water-injected control oocytes. These findings suggest that flufenamate via interaction with fNaDC-3 increased the K+ conductance. Acetylsalicylate, indomethacin, and salicylate showed small potential-dependent inward currents in fNaDC-3 but not in hNaDC-3 expressing oocytes. Benzylpenicillin induced voltage-dependent inward currents which were Na(+)-dependent in oocytes expressing fNaDC-3. The currents were, however, much smaller than those induced by succinate, reflecting probably a low fit of the monovalent benzylpenicillin to the dicarboxylate binding site. The data show hitherto unknown effects of monovalent anionic drugs on a transporter for divalent di- and tricarboxylates.     

Thallium ions can replace both sodium and potassium ions in the glutamate transporter excitatory amino acid carrier 1

The excitatory amino acid carrier EAAC1 belongs to a family of glutamate transporters that use the electrochemical transmembrane gradients of sodium and potassium to mediate uphill transport of glutamate into the cell. While the sites of cation interaction with EAAC1 are unknown, two cation binding sites were observed in the crystal structure of the bacterial glutamate transporter homologue GltPh. Although occupied by Tl(+) in the crystal structure, these sites were proposed to be Na(+) binding sites. Therefore, we tested whether Tl(+) has the ability to replace Na(+) also in the mammalian transporters. Our data demonstrate that Tl(+) can bind to EAAC1 with high affinity and mediate a host of different functions. Tl(+) can functionally replace potassium when applied to the cytoplasm and can support glutamate transport current. When applied extracellularly, Tl(+) induces some behavior that mimics that of the Na(+)-bound transporter, such as activation of the cation-induced anion conductance and creation of a substrate binding site, but it cannot replace Na(+) in supporting glutamate transport current. Moreover, our data show a differential effect of mutations to two acidic amino acids potentially involved in cation binding (D367 and D454) on Na(+) and Tl(+) affinity. Overall, our results demonstrate that the ability of the glutamate transporters to interact with Tl(+) is conserved between GltPh and a mammalian member of the transporter family. However, in contrast to GltPh, which does not bind K(+), Tl(+) is more efficient in mimicking K(+) than Na(+) when interacting with the mammalian protein.     

Structural and functional characteristics of two sodium-coupled dicarboxylate transporters (ceNaDC1 and ceNaDC2) from Caenorhabditis elegans and their relevance to life span

We have cloned and functionally characterized two Na(+)-coupled dicarboxylate transporters, namely ceNaDC1 and ceNaDC2, from Caenorhabditis elegans. These two transporters show significant sequence homology with the product of the Indy gene identified in Drosophila melanogaster and with the Na(+)-coupled dicarboxylate transporters NaDC1 and NaDC3 identified in mammals. In a mammalian cell heterologous expression system, the cloned ceNaDC1 and ceNaDC2 mediate Na(+)-coupled transport of various dicarboxylates. With succinate as the substrate, ceNaDC1 exhibits much lower affinity compared with ceNaDC2. Thus, ceNaDC1 and ceNaDC2 correspond at the functional level to the mammalian NaDC1 and NaDC3, respectively. The nadc1 and nadc2 genes are not expressed at the embryonic stage, but the expression is detectable all through the early larva stage to the adult stage. Tissue-specific expression pattern studies using a reporter gene fusion approach in transgenic C. elegans show that both genes are coexpressed in the intestinal tract, an organ responsible for not only the digestion and absorption of nutrients but also for the storage of energy in this organism. Independent knockdown of the function of these two transporters in C. elegans using the strategy of RNA interference suggests that NaDC1 is not associated with the regulation of average life span in this organism, whereas the knockdown of NaDC2 function leads to a significant increase in the average life span. Disruption of the function of the high affinity Na(+)-coupled dicarboxylate transporter NaDC2 in C. elegans may lead to decreased availability of dicarboxylates for cellular production of metabolic energy, thus creating a biological state similar to that of caloric restriction, and consequently leading to life span extension.     

Cloning of the human thiamine transporter, a member of the folate transporter family.

We have isolated a cDNA from human placenta, which, when expressed heterologously in mammalian cells, mediates the transport of the water-soluble vitamin thiamine. The cDNA codes for a protein of 497 amino acids containing 12 putative transmembrane domains. Northern blot analysis indicates that this transporter is widely expressed in human tissues. When expressed in HeLa cells, the cDNA induces the transport of thiamine (K(t) = 2.5 +/- 0.6 microM) in a Na(+)-independent manner. The cDNA-mediated transport of thiamine is stimulated by an outwardly directed H(+) gradient. Substrate specificity assays indicate that the transporter is specific to thiamine. Even though thiamine is an organic cation, the cDNA-induced thiamine transport is not inhibited by other organic cations. Similarly, thiamine is not a substrate for the known members of mammalian organic cation transporter family. The thiamine transporter gene, located on human chromosome 1q24, consists of 6 exons and is most likely the gene defective in the metabolic disorder, thiamine-responsive megaloblastic anemia. At the level of amino acid sequence, the thiamine transporter is most closely related to the reduced-folate transporter and thus represents the second member of the folate transporter family.     

Human Na+ -coupled citrate transporter: primary structure,genomic organization,and transport function

This paper describes the cloning and functional characterization of the human Na(+)-coupled citrate transporter (NaCT). The cloned human NaCT shows 77% sequence identity with rat NaCT. The nact gene is located on human chromosome 17 at p12-13. NaCT mRNA is expressed most predominantly in the liver, with moderate expression detectable in the brain and testis. When functionally expressed in mammalian cells, human NaCT mediates the Na(+)-coupled transport of citrate. Studies with several monocarboxylates, dicarboxylates, and tricarboxylates show that the transporter is selective for citrate with comparatively several-fold lower affinity for other intermediates of citric acid cycle. The Michelis-Menten constant for citrate is approximately 650 microM. The activation of citrate transport by Na(+) is sigmoidal, suggesting involvement of multiple Na(+) ions in the activation process. The transport process is electrogenic. This represents the first plasma membrane transporter in humans that mediates the preferential entry of citrate into cells. Citrate occupies a pivotal position in many important biochemical pathways. Among various citric acid cycle intermediates, citrate is present at the highest concentrations in human blood. The selectivity of NaCT towards citrate and its predominant expression in the liver suggest that this transporter may facilitate the utilization of circulating citrate for the generation of metabolic energy and for the synthesis of fatty acids and cholesterol.     

Cloning of a Na(+)- and Cl(-)-dependent betaine transporter that is regulated by hypertonicity.

Many hypertonic bacteria, plants, marine animals, and the mammalian renal medulla are protected from the deleterious effects of high intracellular concentrations of electrolytes by accumulating high concentrations of the nonperturbing osmolyte betaine. When kidney-derived Madin-Darby canine kidney (MDCK) cells are cultured in hypertonic medium, they accumulate betaine to 1,000 times its medium concentration. This results from induction by hypertonicity of high rates of betaine transport into cells. We have isolated a cDNA (BGT-1) encoding a renal betaine transporter by screening an MDCK cell cDNA library for expression of a betaine transporter in Xenopus oocytes. The cDNA encodes a single protein of 614 amino acids, with an estimated molecular weight of 69 kDa. The deduced amino acid sequence exhibits highly significant sequence and topographic similarity to brain gamma-amino-n-butyric acid (GABA) and noradrenaline transporters, suggesting that the renal BGT-1 is a member of the brain GABA/noradrenaline transporter gene family. Expression in oocytes indicates that the BGT-1 protein has both betaine and GABA transport activities that are Cl(-)- as well as Na(+)-dependent and functionally similar to betaine and GABA transport in MDCK cells. Northern hybridization indicates that transporter mRNA is localized to the kidney medulla and is induced in MDCK cells by hypertonicity.     

Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae

The SLC13 transporter family, whose members play key physiological roles in the regulation of fatty acid synthesis, adiposity, insulin resistance, and other processes, catalyzes the transport of Krebs cycle intermediates and sulfate across the plasma membrane of mammalian cells. SLC13 transporters are part of the divalent anion:Na⁺ symporter (DASS) family that includes several well-characterized bacterial members. Despite sharing significant sequence similarity, the functional characteristics of DASS family members differ with regard to their substrate and coupling ion dependence. The publication of a high resolution structure of dimer VcINDY, a bacterial DASS family member, provides crucial structural insight into this transporter family. However, marrying this structural insight to the current functional understanding of this family also demands a comprehensive analysis of the transporter’s functional properties. To this end, we purified VcINDY, reconstituted it into liposomes, and determined its basic functional characteristics. Our data demonstrate that VcINDY is a high affinity, Na⁺-dependent transporter with a preference for C₄- and C₅-dicarboxylates. Transport of the model substrate, succinate, is highly pH dependent, consistent with VcINDY strongly preferring the substrate’s dianionic form. VcINDY transport is electrogenic with succinate coupled to the transport of three or more Na⁺ ions. In contrast to succinate, citrate, bound in the VcINDY crystal structure (in an inward-facing conformation), seems to interact only weakly with the transporter in vitro. These transport properties together provide a functional framework for future experimental and computational examinations of the VcINDY transport mechanism.     

Characterization of the Pho89 phosphate transporter by functional hyperexpression in Saccharomyces cerevisiae

The Na(+)-coupled, high-affinity Pho89 plasma membrane phosphate transporter in Saccharomyces cerevisiae has so far been difficult to study because of its low activity and special properties. In this study, we have used a pho84Deltapho87Deltapho90Deltapho91Delta quadruple deletion strain of S. cerevisiae devoid of all transporter genes specific for inorganic phosphate, except for PHO89, to functionally characterize Pho89 under conditions where its expression is hyperstimulated. Under these conditions, the Pho89 protein is strongly upregulated and is the sole high-capacity phosphate transporter sustaining cellular acquisition of inorganic phosphate. Even if Pho89 is synthesized in cells grown at pH 4.5-8.0, the transporter is functionally active under alkaline conditions only, with a K(m) value reflecting high-affinity properties of the transporter and with a transport rate about 100-fold higher than that of the protein in a wild-type strain. Even under these hyperexpressive conditions, Pho89 is unable to sense and signal extracellular phosphate levels. In cells grown at pH 8.0, Pho89-mediated phosphate uptake at alkaline pH is cation-dependent with a strong activation by Na(+) ions and sensitivity to carbonyl cyanide m-chlorophenylhydrazone. The contribution of H(+)- and Na(+)-coupled phosphate transport systems in wild-type cells grown at different pH values was quantified. The contribution of the Na(+)-coupled transport system to the total cellular phosphate uptake activity increases progressively with increasing pH.     

A hyperosmotic stress-induced mRNA of carp cell encodes Na(+)- and Cl(-)-dependent high affinity taurine transporter

A cDNA clone encoding a Na(+)- and Cl(-)-dependent high affinity taurine transporter was isolated from a common carp cell line, Epithelioma papulosum cyprini (EPC), as a hyperosmotic stress-inducible gene by RNA arbitrarily primed PCR. The clone contained a 2.5-kb cDNA fragment including an open reading frame of 1878 bp encoding a protein of 625 amino acids. The deduced amino acid sequence of carp taurine transporter shows 78-80% identity to those of cloned mammalian taurine transporters. The functional characteristics of the cloned transporter were analyzed by expression in COS-7 cells. Transfection with the cDNA induced Na(+)- and Cl(-)-dependent taurine transport activity with an apparent K(m) of 56 microM. The Na(+)/Cl(-)hepatopancreas. Taurine transporter mRNA level increased up to 7.5-fold on raising the ambient osmolality from 300 to 450 mosmol/kgH(2)O. These data suggest the significant role of taurine as an osmolyte in carp cells.     

Bicarbonate transport proteins.

Bicarbonate is not freely permeable to membranes. Yet, bicarbonate must be moved across membranes, as part of CO2 metabolism and to regulate cell pH. Mammalian cells ubiquitously express bicarbonate transport proteins to facilitate the transmembrane bicarbonate flux. These bicarbonate transporters, which function by different transport mechanisms, together catalyse transmembrane bicarbonate movement. Recent advances have allowed the identification of several new bicarbonate transporter genes. Bicarbonate transporters cluster into two separate families: (i) the anion exachanger (AE) family of Cl-/HCO3- exchangers is related in sequence to the NBC family of Na+/HCO3- cotransporters and the Na(+)-dependent Cl/HCO3- exchangers and (ii) some members of the SLC26a family of sulfate transporters will also transport bicarbonate but are not related in sequence to the AE/NBC family of transporters. This review summarizes our understanding of the mammalian bicarbonate transporter superfamily.     

Mechanistic insights and functional determinants of the transport cycle of the ascorbic acid transporter SVCT2. Activation by sodium and absolute dependence on bivalent cations

We characterized the human Na(+)-ascorbic acid transporter SVCT2 and developed a basic model for the transport cycle that challenges the current view that it functions as a Na(+)-dependent transporter. The properties of SVCT2 are modulated by Ca(2+)/Mg(2+) and a reciprocal functional interaction between Na(+) and ascorbic acid that defines the substrate binding order and the transport stoichiometry. Na(+) increased the ascorbic acid transport rate in a cooperative manner, decreasing the transport K(m) without affecting the V(max), thus converting a low affinity form of the transporter into a high affinity transporter. Inversely, ascorbic acid affected in a bimodal and concentration-dependent manner the Na(+) cooperativity, with absence of cooperativity at low and high ascorbic acid concentrations. Our data are consistent with a transport cycle characterized by a Na(+):ascorbic acid stoichiometry of 2:1 and a substrate binding order of the type Na(+):ascorbic acid:Na(+). However, SVCT2 is not electrogenic. SVCT2 showed an absolute requirement for Ca(2+)/Mg(2+) for function, with both cations switching the transporter from an inactive into an active conformation by increasing the transport V(max) without affecting the transport K(m) or the Na(+) cooperativity. Our data indicate that SVCT2 may switch between a number of states with characteristic properties, including an inactive conformation in the absence of Ca(2+)/Mg(2+). At least three active states can be envisioned, including a low affinity conformation at Na(+) concentrations below 20 mM and two high affinity conformations at elevated Na(+) concentrations whose Na(+) cooperativity is modulated by ascorbic acid. Thus, SVCT2 is a Ca(2+)/Mg(2+)-dependent transporter.     

Determinants of substrate and cation affinities in the Na+/dicarboxylate cotransporter.

The Na+/dicarboxylate cotransporter (NaDC-1) couples the transport of sodium and tricarboxylic acid cycle intermediates, such as succinate and citrate. The rabbit and human homologues (rbNaDC-1 and hNaDC-1, respectively) are 78% identical in amino acid sequence but exhibit several differences in their functional properties. rbNaDC-1 has a greater apparent affinity for citrate and sodium than hNaDC-1. Furthermore, unlike hNaDC-1, rbNaDC-1 is inhibited by low concentrations of lithium. In this study, chimeric transporters were constructed to identify the protein domains responsible for the functional differences between rbNaDC-1 and hNaDC-1. Individual substitutions of transmembrane domain (TMD) 7, 10 or 11 produced transporters with intermediate properties. However, substitution of TMD 7, 10, and 11 together resulted in a transporter with the citrate Km of the donor, suggesting that interactions between these domains determine the differences in apparent citrate affinities. TMDs 10 and 11 are most important in determining the differences in apparent sodium affinities, and TMD 11 determines the sensitivity to lithium inhibition. We conclude that transmembrane domains 7, 10, and 11 in NaDC-1 may contain at least one of the cation binding sites in close proximity to the substrate binding domain.     

Molecular and functional characterization of a Na(+)-K(+) transporter from the Trk family in the ectomycorrhizal fungus Hebeloma cylindrosporum

Ectomycorrhizal symbiosis between fungi and woody plants strongly improves plant mineral nutrition and constitutes a major biological process in natural ecosystems. Molecular identification and functional characterization of fungal transport systems involved in nutrient uptake are crucial steps toward understanding the improvement of plant nutrition and the symbiotic relationship itself. In the present report a transporter belonging to the Trk family is identified in the model ectomycorrhizal fungus Hebeloma cylindrosporum and named HcTrk1. The Trk family is still poorly characterized, although it plays crucial roles in K(+) transport in yeasts and filamentous fungi. In Saccharomyces cerevisiae K(+) uptake is mainly dependent on the activity of Trk transporters thought to mediate H(+):K(+) symport. The ectomycorrhizal HcTrk1 transporter was functional when expressed in Xenopus oocytes, enabling the first electrophysiological characterization of a transporter from the Trk family. HcTrk1 mediates instantaneously activating inwardly rectifying currents, is permeable to both K(+) and Na(+), and displays channel-like functional properties. The whole set of data and particularly a phenomenon reminiscent of the anomalous mole fraction effect suggest that the transport does not occur according to the classical alternating access model. Permeation appears to occur through a single-file pore, where interactions between Na(+) and K(+) might result in Na(+):K(+) co-transport activity. HcTrk1 is expressed in external hyphae that explore the soil when the fungus grows in symbiotic condition. Thus, it could play a major role in both the K(+) and Na(+) nutrition of the fungus (and of the plant) in nutrient-poor soils.