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1
Milanick, M. A., and R. B. Gunn. "Proton inhibition of chloride exchange: asynchrony of band 3 proton and anion transport sites?" American Journal of Physiology-Cell Physiology 250, no. 6 (June 1, 1986): C955—C969. http://dx.doi.org/10.1152/ajpcell.1986.250.6.c955.
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The inhibition of chloride exchange at 0 degrees C by protons at the cytoplasmic and the extracellular surface of the band 3 protein of human erythrocytes was measured between pH 4.6 and 7.6. At constant external pH and chloride concentration, internal protons were a mixed inhibitor of chloride flux, with the apparent pK2 = 6.1 for protonation of the inward-facing empty transporter conformation and the apparent pK3 = 5.7 for protonation of the chloride-transporter complex. The activation of chloride exchange by external chloride was inhibited by internal protons, and internal protonation of the externally facing empty conformation had a pK1 = 6.1. External protons were also a mixed inhibitor of chloride exchange with the apparent pK1 = 5.0 for the empty outward-facing transporter conformation. Because of the pHo dependence of self-inhibition, the value of pK3 on the outside for chloride could not be accurately determined, but the apparent pK3 for protonation of the iodide-transporter complex on the extracellular surface was 4.9. The data support a mechanism with a single proton binding site that can alternatively have access to the cytoplasmic and extracellular solutions. It appears that this proton binding and transport site can be coupled to the single anion transport site for cotransport, but the two sites can be on opposite sides of the membrane at the same time and thus can be asynchronously transported by conformational changes of band 3.
2
Becker, Holger M., and Joachim W. Deitmer. "Transport Metabolons and Acid/Base Balance in Tumor Cells." Cancers 12, no. 4 (April 7, 2020): 899. http://dx.doi.org/10.3390/cancers12040899.
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Solid tumors are metabolically highly active tissues, which produce large amounts of acid. The acid/base balance in tumor cells is regulated by the concerted interplay between a variety of membrane transporters and carbonic anhydrases (CAs), which cooperate to produce an alkaline intracellular, and an acidic extracellular, environment, in which cancer cells can outcompete their adjacent host cells. Many acid/base transporters form a structural and functional complex with CAs, coined “transport metabolon”. Transport metabolons with bicarbonate transporters require the binding of CA to the transporter and CA enzymatic activity. In cancer cells, these bicarbonate transport metabolons have been attributed a role in pH regulation and cell migration. Another type of transport metabolon is formed between CAs and monocarboxylate transporters, which mediate proton-coupled lactate transport across the cell membrane. In this complex, CAs function as “proton antenna” for the transporter, which mediate the rapid exchange of protons between the transporter and the surroundings. These transport metabolons do not require CA catalytic activity, and support the rapid efflux of lactate and protons from hypoxic cancer cells to allow sustained glycolytic activity and cell proliferation. Due to their prominent role in tumor acid/base regulation and metabolism, transport metabolons might be promising drug targets for new approaches in cancer therapy.
3
Sacher, A., A. Cohen, and N. Nelson. "Properties of the mammalian and yeast metal-ion transporters DCT1 and Smf1p expressed in Xenopus laevis oocytes." Journal of Experimental Biology 204, no. 6 (March 15, 2001): 1053–61. http://dx.doi.org/10.1242/jeb.204.6.1053.
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Transition metals are essential for many metabolic processes, and their homeostasis is crucial for life. Metal-ion transporters play a major role in maintaining the correct concentrations of the various metal ions in living cells. Little is known about the transport mechanism of metal ions by eukaryotic cells. Some insight has been gained from studies of the mammalian transporter DCT1 and the yeast transporter Smf1p by following the uptake of various metal ions and from electrophysiological experiments using Xenopus laevis oocytes injected with RNA copies (c-RNA) of the genes for these transporters. Both transporters catalyze the proton-dependent uptake of divalent cations accompanied by a ‘slippage’ phenomenon of different monovalent cations unique to each transporter. Here, we further characterize the transport activity of DCT1 and Smf1p, their substrate specificity and their transport properties. We observed that Zn(2+) is not transported through the membrane of Xenopus laevis oocytes by either transporter, even though it inhibits the transport of the other metal ions and enables protons to ‘slip’ through the DCT1 transporter. A special construct (Smf1p-s) was made to enhance Smf1p activity in oocytes to enable electrophysiological studies of Smf1p-s-expressing cells. 54Mn(2+) uptake by Smf1p-s was measured at various holding potentials. In the absence of Na(+) and at pH 5.5, metal-ion uptake was not affected by changes in negative holding potentials. Elevating the pH of the medium to 6.5 caused metal-ion uptake to be influenced by the holding potential: ion uptake increased when the potential was lowered. Na(+) inhibited metal-ion uptake in accordance with the elevation of the holding potential. A novel clutch mechanism of ion slippage that operates via continuously variable stoichiometry between the driving-force pathway (H(+)) and the transport pathway (divalent metal ions) is proposed. The possible physiological advantages of proton slippage through DCT1 and of Na(+) slippage through Smf1p are discussed.
4
Sharma, Neha, Nanda G. Aduri, Anna Iqbal, Bala K. Prabhala, and Osman Mirza. "Peptide Selectivity of the Proton-Coupled Oligopeptide Transporter from Neisseria meningitidis." Journal of Molecular Microbiology and Biotechnology 26, no. 5 (2016): 312–19. http://dx.doi.org/10.1159/000447129.
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Peptide transport in living organisms is facilitated by either primary transport, hydrolysis of ATP, or secondary transport, cotransport of protons. In this study, we focused on investigating the ligand specificity of the <i>Neisseria meningitidis</i> proton-coupled oligopeptide transporter (NmPOT). It has been shown that the gene encoding this transporter is upregulated during infection. NmPOT conformed to the typical chain length preference as observed in prototypical transporters of this family. In contrast to prototypical transporters, it was unable to accommodate a positively charged peptide residue at the C-terminus position of the substrate peptide. Sequence analysis of the active site of NmPOT displayed a distinctive aromatic patch, which has not been observed in any other transporters from this family. This aromatic patch may be involved in providing NmPOT with its atypical preferences. This study provides important novel information towards understanding how these transporters recognize their substrates.
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Cheeseman, Chris. "GLUT7: a new intestinal facilitated hexose transporter." American Journal of Physiology-Endocrinology and Metabolism 295, no. 2 (August 2008): E238—E241. http://dx.doi.org/10.1152/ajpendo.90394.2008.
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The very last member of the SLC2A gene family of facilitated hexose transporters to be cloned was SLC2A7 (hGLUT7). It has been assigned to the class II of the GLUT family on the basis of sequence similarity, and its closest family member is GLUT5, an intestinal fructose transporter. GLUT7 is primarily expressed in the small intestine and colon, although mRNA has been detected in the testis and prostate as well. The protein is expressed in the apical membrane of the small intestine and colon, and it has a high affinity (<0.5 mM) for glucose and fructose. The abundance of the protein in the small intestine does change in parallel with the dietary carbohydrate. However, the distribution of GLUT7 along the small intestine does not entirely match with the availability of glucose and fructose, suggesting that the physiological substrate for this transporter has yet to be identified. Unlike GLUT13, the proton-coupled myoinositol transporter (HMIT), there is no evidence for the coupling of protons to the hexose movement via GLUT7. One area of study in which GLUT7 has provided a useful comparison with GLUT1 has been in the development of the hypothesis that the facilitated hexose transporters may have a selectivity filter at the exofacial opening of the translocation pore, which helps to determine which hexoses can be transported. If substantiated, the elucidation of this mechanism may prove useful in the design of hexose analogs for use in cancer imaging and therapeutics.
6
Adibi, SA. "Intestinal Oligopeptide Transporter: From Hypothesis to Cloning." Physiology 11, no. 3 (June 1, 1996): 133–37. http://dx.doi.org/10.1152/physiologyonline.1996.11.3.133.
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Several decades ago it was hypothesized that there is an active intestinal oligopeptide transporter that allows large-scale absorption of dipeptides and tripeptides. After a period of uncertainty, this hypothesis was recently validated by the cloning of an intestinal oligopeptide transporter that appears to belong to a superfamily of proton-dependent transporters.
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Groeneveld, Maarten, Ruud G. J. Detert Oude Weme, Ria H. Duurkens, and Dirk Jan Slotboom. "Biochemical Characterization of the C4-Dicarboxylate Transporter DctA from Bacillus subtilis." Journal of Bacteriology 192, no. 11 (April 2, 2010): 2900–2907. http://dx.doi.org/10.1128/jb.00136-10.
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ABSTRACT Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C4-dicarboxylates. Here, we have expressed the DctA homologue from Bacillus subtilis in the Gram-positive bacterium Lactococcus lactis. Transport of dicarboxylates in vitro in isolated membrane vesicles was assayed. We determined the substrate specificity, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns. DctA was found to catalyze proton-coupled symport of the four C4-dicarboxylates from the Krebs cycle (succinate, fumurate, malate, and oxaloacetate) but not of other mono- and dicarboxylates. Because (i) succinate-proton symport was electrogenic (stimulated by an internal negative membrane potential) and (ii) the divalent anionic form of succinate was recognized by DctA, at least three protons must be cotransported with succinate. The results were interpreted in the light of the crystal structure of the homologous aspartate transporter GltPh from Pyrococcus horikoshii.
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Dastvan, Reza, Axel W. Fischer, Smriti Mishra, Jens Meiler, and Hassane S. Mchaourab. "Protonation-dependent conformational dynamics of the multidrug transporter EmrE." Proceedings of the National Academy of Sciences 113, no. 5 (January 19, 2016): 1220–25. http://dx.doi.org/10.1073/pnas.1520431113.
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The small multidrug transporter from Escherichia coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient. Although principal mechanistic elements of proton/substrate antiport have been described, the structural record is limited to the conformation of the substrate-bound state, which has been shown to undergo isoenergetic alternating access. A central but missing link in the structure/mechanism relationship is a description of the proton-bound state, which is an obligatory intermediate in the transport cycle. Here we report a systematic spin labeling and double electron electron resonance (DEER) study that uncovers the conformational changes of EmrE subsequent to protonation of critical acidic residues in the context of a global description of ligand-induced structural rearrangements. We find that protonation of E14 leads to extensive rotation and tilt of transmembrane helices 1–3 in conjunction with repacking of loops, conformational changes that alter the coordination of the bound substrate and modulate its access to the binding site from the lipid bilayer. The transport model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers alternating access between inward- and outward-facing conformations of the substrate-loaded transporter, thus enabling antiport without dissipation of the proton gradient.
9
Sigal, Nadejda, Shahar Molshanski-Mor, and Eitan Bibi. "No Single Irreplaceable Acidic Residues in the Escherichia coli Secondary Multidrug Transporter MdfA." Journal of Bacteriology 188, no. 15 (August 1, 2006): 5635–39. http://dx.doi.org/10.1128/jb.00422-06.
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ABSTRACT The largest family of solute transporters (major facilitator superfamily [MFS]) includes proton-motive-force-driven secondary transporters. Several characterized MFS transporters utilize essential acidic residues that play a critical role in the energy-coupling mechanism during transport. Surprisingly, we show here that no single acidic residue plays an irreplaceable role in the Escherichia coli secondary multidrug transporter MdfA.
10
Zulkifli, Mohammad, and Anand Kumar Bachhawat. "Identification of residues critical for proton-coupled glutathione translocation in the yeast glutathione transporter, Hgt1p." Biochemical Journal 474, no. 11 (May 16, 2017): 1807–21. http://dx.doi.org/10.1042/bcj20161063.
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The proton gradient acts as the driving force for the transport of many metabolites across fungal and plant plasma membranes. Identifying the mechanism of proton relay is critical for understanding the mechanism of transport mediated by these transporters. We investigated two strategies for identifying residues critical for proton-dependent substrate transport in the yeast glutathione transporter, Hgt1p, a member of the poorly understood oligopeptide transporter family of transporters. In the first strategy, we tried to identify the pH-independent mutants that could grow at higher pH when dependant on glutathione transport. Screening a library of 269 alanine mutants of the transmembrane domains (TMDs) along with a random mutagenesis strategy yielded two residues (E135K on the cusp of TMD2 and N710S on TMD12) that permitted growth on glutathione at pH 8.0. Further analysis revealed that these residues were not involved in proton symport even though they conferred better transport at a higher pH. The second strategy involved a knowledge-driven approach, targeting 31 potential residues based on charge, conservation and location. Mutation of these residues followed by functional and biochemical characterization revealed E177A, Y193A, D335A, Y374A, H445A and R554A as being defective in proton transport. Further analysis enabled possible roles of these residues to be assigned in proton relay. The implications of these findings in relation to Hgt1p and the suitability of these strategic approaches for identifying such residues are discussed.
11
Li, Bin, Kaiduan Zhang, Yong Nie, Xianping Wang, Yan Zhao, Xuejun C. Zhang, and Xiao-Lei Wu. "Structure of theDietziaMrp complex reveals molecular mechanism of this giant bacterial sodium proton pump." Proceedings of the National Academy of Sciences 117, no. 49 (November 23, 2020): 31166–76. http://dx.doi.org/10.1073/pnas.2006276117.
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Multiple resistance and pH adaptation (Mrp) complexes are sophisticated cation/proton exchangers found in a vast variety of alkaliphilic and/or halophilic microorganisms, and are critical for their survival in highly challenging environments. This family of antiporters is likely to represent the ancestor of cation pumps found in many redox-driven transporter complexes, including the complex I of the respiratory chain. Here, we present the three-dimensional structure of the Mrp complex from aDietziasp. strain solved at 3.0-Å resolution using the single-particle cryoelectron microscopy method. Our structure-based mutagenesis and functional analyses suggest that the substrate translocation pathways for the driving substance protons and the substrate sodium ions are separated in two modules and that symmetry-restrained conformational change underlies the functional cycle of the transporter. Our findings shed light on mechanisms of redox-driven primary active transporters, and explain how driving substances of different electric charges may drive similar transport processes.
12
Parker, Joanne L., Chenghan Li, Allete Brinth, Zhi Wang, Lutz Vogeley, Nicolae Solcan, Gregory Ledderboge-Vucinic, et al. "Proton movement and coupling in the POT family of peptide transporters." Proceedings of the National Academy of Sciences 114, no. 50 (November 27, 2017): 13182–87. http://dx.doi.org/10.1073/pnas.1710727114.
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POT transporters represent an evolutionarily well-conserved family of proton-coupled transport systems in biology. An unusual feature of the family is their ability to couple the transport of chemically diverse ligands to an inwardly directed proton electrochemical gradient. For example, in mammals, fungi, and bacteria they are predominantly peptide transporters, whereas in plants the family has diverged to recognize nitrate, plant defense compounds, and hormones. Although recent structural and biochemical studies have identified conserved sites of proton binding, the mechanism through which transport is coupled to proton movement remains enigmatic. Here we show that different POT transporters operate through distinct proton-coupled mechanisms through changes in the extracellular gate. A high-resolution crystal structure reveals the presence of ordered water molecules within the peptide binding site. Multiscale molecular dynamics simulations confirm proton transport occurs through these waters via Grotthuss shuttling and reveal that proton binding to the extracellular side of the transporter facilitates a reorientation from an inward- to outward-facing state. Together these results demonstrate that within the POT family multiple mechanisms of proton coupling have likely evolved in conjunction with variation of the extracellular gate.
13
Juel, C. "Lactate-proton cotransport in skeletal muscle." Physiological Reviews 77, no. 2 (April 1, 1997): 321–58. http://dx.doi.org/10.1152/physrev.1997.77.2.321.
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Skeletal muscle and most other tissues possess a membrane transport system mediating a coupled lactate and H+ translocation. Muscle possesses several lactate-proton transporter isoforms of which two have been cloned; however, the main isoform remains to be identified. The isoforms may have different properties and functional roles, but these have not been specifically characterized. The distribution of lactate-proton transport capacity in skeletal muscle is fiber type dependent, with a higher capacity in slow-twitch fibers compared with fast-twitch fibers. During intense muscle activity and in the recovery period, the lactate and H+ effluxes are mainly mediated by the lactate-proton transporter, which reduces the accumulation of lactate in muscle as well as the drop in internal pH suggested to be involved in muscle fatigue. Thus the lactate-proton transporter is of functional importance for pH regulation in association with muscle activity. This carrier is also important for lactate uptake into resting muscle and other tissues; therefore, the carrier distribution is important for the fate of lactate in the body. In addition, the capacity of the lactate-proton transporter can be increased by intense training and is reduced by inactivity; thus the lactate-proton transporter can undergo adaptive changes.
14
Newstead, Simon. "Towards a structural understanding of drug and peptide transport within the proton-dependent oligopeptide transporter (POT) family." Biochemical Society Transactions 39, no. 5 (September 21, 2011): 1353–58. http://dx.doi.org/10.1042/bst0391353.
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One of the principal aims of modern drug design is the targeted delivery of drugs within the body, such as to the central nervous system, combined with their exclusion from the liver and kidneys, which break down foreign molecules and subsequently eliminate them. Many of the commonly prescribed drugs are transported into cells and across the plasma membrane via endogenous membrane transporters, whose principal roles are the uptake of essential nutrients for metabolism. In many cases, such drug transport is serendipitous as they are simply mistaken as ‘natural’ compounds. Many of these transporters could, however, be targeted more efficiently, improving drug absorption, distribution and retention. The molecular details of these drug–transporter interactions, however, are at best poorly understood, in large part through the absence of any high-resolution structural information. To address this issue, we recently determined the structure of a prokaryotic peptide transporter, PepTSo from Shewanella oneidensis, which shares a high degree of sequence similarity and functional characteristics with the human PepT1 and PepT2 proteins. PepT1 and PepT2 contribute significantly to the oral bioavailability and pharmacokinetic properties of a number of important drug families, including antibiotics, antivirals and anticancer agents. The crystal structure of PepTSo provides the first high-resolution model of a drug importer and provides the starting point for understanding drug and peptide transport within the human body.
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McDermott, Joseph R., Barry P. Rosen, and Zijuan Liu. "Jen1p: A High Affinity Selenite Transporter in Yeast." Molecular Biology of the Cell 21, no. 22 (November 15, 2010): 3934–41. http://dx.doi.org/10.1091/mbc.e10-06-0513.
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Selenium is a micronutrient in most eukaryotes, including humans, which is well known for having an extremely thin border between beneficial and toxic concentrations. Soluble tetravalent selenite is the predominant environmental form and also the form that is applied in the treatment of human diseases. To acquire this nutrient from low environmental concentrations as well as to avoid toxicity, a well-controlled transport system is required. Here we report that Jen1p, a proton-coupled monocarboxylate transporter in S. cerevisiae, catalyzes high-affinity uptake of selenite. Disruption of JEN1 resulted in selenite resistance, and overexpression resulted in selenite hypersensitivity. Transport assay showed that overexpression of Jen1p enables selenite accumulation in yeast compared with a JEN1 knock out strain, indicating the Jen1p transporter facilitates selenite accumulation inside cells. Selenite uptake by Jen1p had a Kmof 0.91 mM, which is comparable to the Kmfor lactate. Jen1p transported selenite in a proton-dependent manner which resembles the transport mechanism for lactate. In addition, selenite and lactate can inhibit the transport of each other competitively. Therefore, we postulate selenite is a molecular mimic of monocarboxylates which allows selenite to be transported by Jen1p.
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Takatsuka, Yumiko, and Hiroshi Nikaido. "Threonine-978 in the Transmembrane Segment of the Multidrug Efflux Pump AcrB of Escherichia coli Is Crucial for Drug Transport as a Probable Component of the Proton Relay Network." Journal of Bacteriology 188, no. 20 (October 1, 2006): 7284–89. http://dx.doi.org/10.1128/jb.00683-06.
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ABSTRACT Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals and expels them from cells. It is a proton antiporter belonging to the resistance-nodulation-division (RND) superfamily. Asp407, Asp408, Lys940, and Arg971 in transmembrane (TM) helices of this transporter have been identified as essential amino acid residues that probably function as components of the proton relay system. In this study, we identified a novel residue in TM helix 11, Thr978, as an essential residue by alanine scanning mutagenesis. Its location close to Asp407 suggests that it is also a component of the proton translocation pathway, a prediction confirmed by the similar conformations adopted by T978A, D407A, D408A, and K940A mutant proteins (see the accompanying paper). Sequence alignment of 566 RND transporters showed that this threonine residue is conserved in about 96% of cases. Our results suggest the hypotheses that Thr978 functions through hydrogen bonding with Asp407 and that protonation of the latter alters the salt bridging and hydrogen bonding pattern in the proton relay network, thus initiating a series of conformational changes that ultimately result in drug extrusion.
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Bader, Annika, and Eric Beitz. "Transmembrane Facilitation of Lactate/H+ Instead of Lactic Acid Is Not a Question of Semantics but of Cell Viability." Membranes 10, no. 9 (September 15, 2020): 236. http://dx.doi.org/10.3390/membranes10090236.
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Transmembrane transport of monocarboxylates is conferred by structurally diverse membrane proteins. Here, we describe the pH dependence of lactic acid/lactate facilitation of an aquaporin (AQP9), a monocarboxylate transporter (MCT1, SLC16A1), and a formate–nitrite transporter (plasmodium falciparum FNT, PfFNT) in the equilibrium transport state. FNTs exhibit a channel-like structure mimicking the aquaporin-fold, yet act as secondary active transporters. We used radiolabeled lactate to monitor uptake via yeast-expressed AQP9, MCT1, and PfFNT for long enough time periods to reach the equilibrium state in which import and export rates are balanced. We confirmed that AQP9 behaved perfectly equilibrative for lactic acid, i.e., the neutral lactic acid molecule enters and passes the channel. MCT1, in turn, actively used the transmembrane proton gradient and acted as a lactate/H+ co-transporter. PfFNT behaved highly similar to the MCT in terms of transport properties, although it does not adhere to the classical alternating access transporter model. Instead, the FNT appears to use the proton gradient to neutralize the lactate anion in the protein’s vestibule to generate lactic acid in a place that traverses the central hydrophobic transport path. In conclusion, we propose to include FNT-type proteins into a more generalized, function-based transporter definition.
18
Liu, Yu, Chenghan Li, Meghna Gupta, Nidhi Verma, Atul Kumar Johri, Robert M. Stroud, and Gregory A. Voth. "Key computational findings reveal proton transfer as driving the functional cycle in the phosphate transporter PiPT." Proceedings of the National Academy of Sciences 118, no. 25 (June 16, 2021): e2101932118. http://dx.doi.org/10.1073/pnas.2101932118.
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Phosphate is an indispensable metabolite in a wide variety of cells and is involved in nucleotide and lipid synthesis, signaling, and chemical energy storage. Proton-coupled phosphate transporters within the major facilitator family are crucial for phosphate uptake in plants and fungi. Similar proton-coupled phosphate transporters have been found in different protozoan parasites that cause human diseases, in breast cancer cells with elevated phosphate demand, in osteoclast-like cells during bone reabsorption, and in human intestinal Caco2BBE cells for phosphate homeostasis. However, the mechanism of proton-driven phosphate transport remains unclear. Here, we demonstrate in a eukaryotic, high-affinity phosphate transporter from Piriformospora indica (PiPT) that deprotonation of aspartate 324 (D324) triggers phosphate release. Quantum mechanics/molecular mechanics molecular dynamics simulations combined with free energy sampling have been employed here to identify the proton transport pathways from D324 upon the transition from the occluded structure to the inward open structure and phosphate release. The computational insights so gained are then corroborated by studies of D45N and D45E amino acid substitutions via mutagenesis experiments. Our findings confirm the function of the structurally predicted cytosolic proton exit tunnel and suggest insights into the role of the titratable phosphate substrate.
19
Taylor, Peter M. "Amino acid transporters: éminences grises of nutrient signalling mechanisms?" Biochemical Society Transactions 37, no. 1 (January 20, 2009): 237–41. http://dx.doi.org/10.1042/bst0370237.
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Nutrient signalling by the mTOR (mammalian target of rapamycin) pathway involves upstream sensing of free AA (amino acid) concentrations. Several AA-regulated kinases have recently been identified as putative intracellular AA sensors. Their activity will reflect the balance between AA flows through underlying mechanisms which together determine the size of the intracellular free AA pool. For indispensable AAs, these mechanisms are primarily (i) AA transport across the cell membrane, and (ii) protein synthesis/breakdown. The System L AA transporter is the primary conduit for cellular entry of indispensable neutral AAs (including leucine and phenylalanine) and potentially a key modulator of AA-sensitive mTOR signalling. Coupling of substrate flows through System L and other AA transporters (e.g. System A) may extend the scope for sensing nutrient abundance. Factors influencing AA transporter activity (e.g. hormones) may affect intracellular AA concentrations and hence indirectly mTOR pathway activity. Several AA transporters are themselves regulated by AA availability through ‘adaptive regulation’, which may help to adjust the gain of AA sensing. The substrate-binding sites of AA transporters are potentially direct sensors of AA availability at both faces of the cell surface, and there is growing evidence that AA transporters of the SNAT (sodium-coupled neutral AA transporter) and PAT (proton-assisted AA transporter) families may operate, at least under some circumstances, as transporter-like sensors (or ‘transceptors’) upstream of mTOR.
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Sun, S., and G. Xu. "Sugar transport in arbuscular mycorrhizal symbiosis." Canadian Journal of Plant Science 89, no. 2 (March 1, 2009): 257–63. http://dx.doi.org/10.4141/cjps07106.
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In arbuscular mycorrhizal (AM) symbioses, there is a reciprocal nutrient exchange, mainly sugar and phosphate, between partners. Transport of phosphate from fungus to plant has been well characterized, and this aspect of AM symbiosis has been reviewed. This mini-review is specifically devoted to sugar transport from plant to fungus in AM symbiosis and discusses the possible links between sugar transporters and AM-inducible inorganic phosphate (Pi) transporters and plasma membrane proton-ATPases in the arbuscule-cortical cell interface. Exploring the sugar transport mechanisms could further contribute to our understanding of nutrient exchange between the two symbiotic partners. Key words: Arbuscular mycorrhizal symbiosis, sugar flux, sugar transporter, phosphate transporter, plasma membrane, H+-ATPase
21
Desmoulin, Sita Kugel, Zhanjun Hou, Aleem Gangjee, and Larry H. Matherly. "The human proton-coupled folate transporter." Cancer Biology & Therapy 13, no. 14 (December 6, 2012): 1355–73. http://dx.doi.org/10.4161/cbt.22020.
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Parker, Joanne L., Justin C. Deme, Zhiyi Wu, Gabriel Kuteyi, Jiandong Huo, Raymond J. Owens, Philip C. Biggin, Susan M. Lea, and Simon Newstead. "Cryo-EM structure of PepT2 reveals structural basis for proton-coupled peptide and prodrug transport in mammals." Science Advances 7, no. 35 (August 2021): eabh3355. http://dx.doi.org/10.1126/sciadv.abh3355.
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The SLC15 family of proton-coupled solute carriers PepT1 and PepT2 play a central role in human physiology as the principal route for acquiring and retaining dietary nitrogen. A remarkable feature of the SLC15 family is their extreme substrate promiscuity, which has enabled the targeting of these transporters for the improvement of oral bioavailability for several prodrug molecules. Although recent structural and biochemical studies on bacterial homologs have identified conserved sites of proton and peptide binding, the mechanism of peptide capture and ligand promiscuity remains unclear for mammalian family members. Here, we present the cryo–electron microscopy structure of the outward open conformation of the rat peptide transporter PepT2 in complex with an inhibitory nanobody. Our structure, combined with molecular dynamics simulations and biochemical and cell-based assays, establishes a framework for understanding peptide and prodrug recognition within this pharmaceutically important transporter family.
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Venter, H., S. Shahi, L. Balakrishnan, S. Velamakanni, A. Bapna, B. Woebking, and H. W. van Veen. "Similarities between ATP-dependent and ion-coupled multidrug transporters." Biochemical Society Transactions 33, no. 5 (October 26, 2005): 1008–11. http://dx.doi.org/10.1042/bst0331008.
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The movement of drugs across biological membranes is mediated by two major classes of membrane transporters. Primary-active, ABC (ATP-binding cassette) multidrug transporters are dependent on ATP-binding/hydrolysis, whereas secondary-active multidrug transporters are coupled to the proton (or sodium)-motive force that exists across the plasma membrane. Recent work on LmrA, an ABC multidrug transporter in Lactococcus lactis, suggests that primary- and secondary-active multidrug transporters share functional and structural features. Some of these similarities and their implications for the mechanism of transport by ABC multidrug transporters will be discussed.
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Higuchi, Kei, Sathish Sivaprakasam, Souad R. Sennoune, Jiro Ogura, Yangzom D. Bhutia, Ricardo Rueda, Suzette L. Pereira, and Vadivel Ganapathy. "A Proton-Coupled Transport System for β-Hydroxy-β-Methylbutyrate (HMB) in Blood–Brain Barrier Endothelial Cell Line hCMEC/D3." Nutrients 13, no. 9 (September 16, 2021): 3220. http://dx.doi.org/10.3390/nu13093220.
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β-Hydroxy-β-methylbutyrate (HMB), a leucine metabolite, is used as a nutritional ingredient to improve skeletal muscle health. Preclinical studies indicate that this supplement also elicits significant benefits in the brain; it promotes neurite outgrowth and prevents age-related reductions in neuronal dendrites and cognitive performance. As orally administered HMB elicits these effects in the brain, we infer that HMB crosses the blood–brain barrier (BBB). However, there have been no reports detailing the transport mechanism for HMB in BBB. Here we show that HMB is taken up in the human BBB endothelial cell line hCMEC/D3 via H+-coupled monocarboxylate transporters that also transport lactate and β-hydroxybutyrate. MCT1 (monocarboxylate transporter 1) and MCT4 (monocarboxylate transporter 4) belonging to the solute carrier gene family SLC16 (solute carrier, gene family 16) are involved, but additional transporters also contribute to the process. HMB uptake in BBB endothelial cells results in intracellular acidification, demonstrating cotransport with H+. Since HMB is known to activate mTOR with potential to elicit transcriptomic changes, we examined the influence of HMB on the expression of selective transporters. We found no change in MCT1 and MCT4 expression. Interestingly, the expression of LAT1 (system L amino acid transporter 1), a high-affinity transporter for branched-chain amino acids relevant to neurological disorders such as autism, is induced. This effect is dependent on mTOR (mechanistic target of rapamycine) activation by HMB with no involvement of histone deacetylases. These studies show that HMB in systemic circulation can cross the BBB via carrier-mediated processes, and that it also has a positive influence on the expression of LAT1, an important amino acid transporter in the BBB.
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Thamotharan, M., V. Zonno, C. Storelli, and G. A. Ahearn. "Basolateral dipeptide transport by the intestine of the teleost Oreochromis mossambicus." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 270, no. 5 (May 1, 1996): R948—R954. http://dx.doi.org/10.1152/ajpregu.1996.270.5.r948.
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Transport characteristics of [14C]glycylsarcosine ([14C]Gly-Sar) were measured in herbivorous tilapi (Oreochromis mossambicus) intestinal basolateral membrane vesicles (BLMV) purified with Percoll gradient centrifugation. Specific activity of the vesicle Na(+)-K(+)-adenosinetriphos- phatase was increased 12-fold, whereas specific activity of the brush-border enzyme alkaline phosphatase was enriched only by 0.8-fold. [14C]Gly-Sar uptake was stimulated by increasing concentrations of extravesicular protons rather than by a transmembrane proton gradient. A transmembrane K+ diffusion potential (inside negative) did not stimulate [14C]Gly-Sar uptake above that observed with short-circuited vesicles. An inwardly directed Na+ gradient had no effect on peptide uptake. Kinetic analysis of basolateral transport rate revealed that the transport occurred by a saturable process conforming to Michaelis-Menten kinetics [Kt [concentration of [14C]Gly-Sar that yielded one-half of maximal influx (Jmax)] = 13.27 +/- 3.80 mM, Jmax = 15,155 +/- 3,096 pmol.mg protein-1.6 s-1]. The basolateral transporter was insensitive to diethylpyrocarbonate (DEP), a specific inhibitor of proton-coupled peptide transport systems. [14C]Gly-Sar influx into tilapia BLMV showed cis-inhibition by several other dipeptides, suggesting that the [14C]Gly-Sar transporter was shared by other peptides too. These observations strongly suggest that the basolateral intestinal dipeptide transporter in herbivorous fishes is distinctly different from either the high- or low-affinity brush-border transporter. It is proton dependent, electroneutral, sodium independent and accepts a wide variety of dipeptides.
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Agboh, Kelvin, Calvin H. F. Lau, Yvonne S. K. Khoo, Himansha Singh, Sagar Raturi, Asha V. Nair, Julie Howard, et al. "Powering the ABC multidrug exporter LmrA: How nucleotides embrace the ion-motive force." Science Advances 4, no. 9 (September 2018): eaas9365. http://dx.doi.org/10.1126/sciadv.aas9365.
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LmrA is a bacterial ATP-binding cassette (ABC) multidrug exporter that uses metabolic energy to transport ions, cytotoxic drugs, and lipids. Voltage clamping in a Port-a-Patch was used to monitor electrical currents associated with the transport of monovalent cationic HEPES+by single-LmrA transporters and ensembles of transporters. In these experiments, one proton and one chloride ion are effluxed together with each HEPES+ion out of the inner compartment, whereas two sodium ions are transported into this compartment. Consequently, the sodium-motive force (interior negative and low) can drive this electrogenic ion exchange mechanism in cells under physiological conditions. The same mechanism is also relevant for the efflux of monovalent cationic ethidium, a typical multidrug transporter substrate. Studies in the presence of Mg-ATP (adenosine 5′-triphosphate) show that ion-coupled HEPES+transport is associated with ATP-bound LmrA, whereas ion-coupled ethidium transport requires ATP binding and hydrolysis. HEPES+is highly soluble in a water-based environment, whereas ethidium has a strong preference for residence in the water-repelling plasma membrane. We conclude that the mechanism of the ABC transporter LmrA is fundamentally related to that of an ion antiporter that uses extra steps (ATP binding and hydrolysis) to retrieve and transport membrane-soluble substrates from the phospholipid bilayer.
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Becker, Holger M. "Carbonic anhydrase IX and acid transport in cancer." British Journal of Cancer 122, no. 2 (December 10, 2019): 157–67. http://dx.doi.org/10.1038/s41416-019-0642-z.
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AbstractAlterations in tumour metabolism and acid/base regulation result in the formation of a hostile environment, which fosters tumour growth and metastasis. Acid/base homoeostasis in cancer cells is governed by the concerted interplay between carbonic anhydrases (CAs) and various transport proteins, which either mediate proton extrusion or the shuttling of acid/base equivalents, such as bicarbonate and lactate, across the cell membrane. Accumulating evidence suggests that some of these transporters interact both directly and functionally with CAIX to form a protein complex coined the ‘transport metabolon’. Transport metabolons formed between bicarbonate transporters and CAIX require CA catalytic activity and have a function in cancer cell migration and invasion. Another type of transport metabolon is formed by CAIX and monocarboxylate transporters. In this complex, CAIX functions as a proton antenna for the transporter, which drives the export of lactate and protons from the cell. Since CAIX is almost exclusively expressed in cancer cells, these transport metabolons might serve as promising targets to interfere with tumour pH regulation and energy metabolism. This review provides an overview of the current state of research on the function of CAIX in tumour acid/base transport and discusses how CAIX transport metabolons could be exploited in modern cancer therapy.
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Subramanian, Veedamali S., Jonathan S. Marchant, and Hamid M. Said. "Apical membrane targeting and trafficking of the human proton-coupled transporter in polarized epithelia." American Journal of Physiology-Cell Physiology 294, no. 1 (January 2008): C233—C240. http://dx.doi.org/10.1152/ajpcell.00468.2007.
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The human proton-coupled folate transporter (hPCFT) is a recently discovered intestinal transporter involved in folate uptake in epithelia (and possibly other cells). Little is currently known about the structure-function relationship of the different domains of this transporter, particularly which regions are important for substrate transport as well as targeting of the transporter to the apical cell surface of polarized cells. Here we have investigated the role of the COOH-terminal domain and a well-conserved sequence separating transmembrane (TM) domains TM2 and TM3 (DXXGRR; amino acids 109–114) speculated by others to be important for transport function. Using live cell imaging approaches, we show that 1) an hPCFT-yellow fluorescent protein construct is functionally expressed at the apical membrane domain and is localized differentially to the human reduced folate carrier; 2) the predicted cytoplasmic COOH-terminal region of hPCFT is not essential for apical targeting or transporter functionality; 3) mutations that ablate a consensus β-turn sequence separating predicted TM2 and TM3 abolished apical [3H]folic acid uptake as a consequence of endoplasmic reticulum retention of mutant, likely misfolded, transporters; and 4) cell surface delivery of hPCFT is disrupted by microtubule depolymerization or by overexpression of the dynactin complex dynamitin (p50). For the first time, our data present information regarding structure-function and membrane targeting of the hPCFT polypeptide, as well as the mechanisms that control its steady-state expression in polarized cells.
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Wang, Hong, You-Jun Fei, Vadivel Ganapathy, and Frederick H. Leibach. "Electrophysiological characteristics of the proton-coupled peptide transporter PEPT2 cloned from rat brain." American Journal of Physiology-Cell Physiology 275, no. 4 (October 1, 1998): C967—C975. http://dx.doi.org/10.1152/ajpcell.1998.275.4.c967.
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We have cloned a peptide transporter from rat brain and found it to be identical to rat kidney PEPT2. In the present study we characterize the transport function of the rat brain PEPT2, with special emphasis on electrophysiological properties and interaction with N-acetyl-l-aspartyl-l-glutamate (NAAG). When heterologously expressed in HeLa cells and in SK-N-SH cells, PEPT2 transports several dipeptides but not free amino acids in the presence of a proton gradient. NAAG competes with other peptides for the PEPT2-mediated transport process. When PEPT2 is expressed in Xenopus laevis oocytes, substrate-induced inward currents are detectable with dipeptides of differing charge in the presence of a proton gradient. Proton activation kinetics are similar for differently charged peptides. NAAG is a transportable substrate for PEPT2, as evidenced by NAAG-induced currents. The Hill coefficient for protons for the activation of the transport of differently charged peptides, including NAAG, is 1. Although the peptide-to-proton stoichiometry for negatively charged peptides is 1, the transport nonetheless is associated with transfer of positive charge into the oocyte, as indicated by peptide-induced inward currents.
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Alekov, Alexi K., and Christoph Fahlke. "Channel-like slippage modes in the human anion/proton exchanger ClC-4." Journal of General Physiology 133, no. 5 (April 13, 2009): 485–96. http://dx.doi.org/10.1085/jgp.200810155.
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The ClC family encompasses two classes of proteins with distinct transport functions: anion channels and transporters. ClC-type transporters usually mediate secondary active anion–proton exchange. However, under certain conditions they assume slippage mode behavior in which proton and anion transport are uncoupled, resulting in passive anion fluxes without associated proton movements. Here, we use patch clamp and intracellular pH recordings on transfected mammalian cells to characterize exchanger and slippage modes of human ClC-4, a member of the ClC transporter branch. We found that the two transport modes differ in transport mechanisms and transport rates. Nonstationary noise analysis revealed a unitary transport rate of 5 × 105 s−1 at +150 mV for the slippage mode, indicating that ClC-4 functions as channel in this mode. In the exchanger mode, unitary transport rates were 10-fold lower. Both ClC-4 transport modes exhibit voltage-dependent gating, indicating that there are active and non-active states for the exchanger as well as for the slippage mode. ClC-4 can assume both transport modes under all tested conditions, with exchanger/channel ratios determined by the external anion. We propose that binding of transported anions to non-active states causes transition from slippage into exchanger mode. Binding and unbinding of anions is very rapid, and slower transitions of liganded and non-liganded states into active conformations result in a stable distribution between the two transport modes. The proposed mechanism results in anion-dependent conversion of ClC-type exchanger into an anion channel with typical attributes of ClC anion channels.
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Youn, Jung-Won, Elena Jolkver, Reinhard Krämer, Kay Marin, and Volker F. Wendisch. "Characterization of the Dicarboxylate Transporter DctA in Corynebacterium glutamicum." Journal of Bacteriology 191, no. 17 (July 6, 2009): 5480–88. http://dx.doi.org/10.1128/jb.00640-09.
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ABSTRACT Transporters of the dicarboxylate amino acid-cation symporter family often mediate uptake of C4-dicarboxylates, such as succinate or l-malate, in bacteria. A member of this family, dicarboxylate transporter A (DctA) from Corynebacterium glutamicum, was characterized to catalyze uptake of the C4-dicarboxylates succinate, fumarate, and l-malate, which was inhibited by oxaloacetate, 2-oxoglutarate, and glyoxylate. DctA activity was not affected by sodium availability but was dependent on the electrochemical proton potential. Efficient growth of C. glutamicum in minimal medium with succinate, fumarate, or l-malate as the sole carbon source required high dctA expression levels due either to a promoter-up mutation identified in a spontaneous mutant or to ectopic overexpression. Mutant analysis indicated that DctA and DccT, a C4-dicarboxylate divalent anion/sodium symporter-type transporter, are the only transporters for succinate, fumarate, and l-malate in C. glutamicum.
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Darbani, Behrooz, Vratislav Stovicek, Steven Axel van der Hoek, and Irina Borodina. "Engineering energetically efficient transport of dicarboxylic acids in yeast Saccharomyces cerevisiae." Proceedings of the National Academy of Sciences 116, no. 39 (August 29, 2019): 19415–20. http://dx.doi.org/10.1073/pnas.1900287116.
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Biobased C4-dicarboxylic acids are attractive sustainable precursors for polymers and other materials. Commercial scale production of these acids at high titers requires efficient secretion by cell factories. In this study, we characterized 7 dicarboxylic acid transporters in Xenopus oocytes and in Saccharomyces cerevisiae engineered for dicarboxylic acid production. Among the tested transporters, the Mae1(p) from Schizosaccharomyces pombe had the highest activity toward succinic, malic, and fumaric acids and resulted in 3-, 8-, and 5-fold titer increases, respectively, in S. cerevisiae, while not affecting growth, which was in contrast to the tested transporters from the tellurite-resistance/dicarboxylate transporter (TDT) family or the Na+ coupled divalent anion–sodium symporter family. Similar to SpMae1(p), its homolog in Aspergillus carbonarius, AcDct(p), increased the malate titer 12-fold without affecting the growth. Phylogenetic and protein motif analyses mapped SpMae1(p) and AcDct(p) into the voltage-dependent slow-anion channel transporter (SLAC1) clade of transporters, which also include plant Slac1(p) transporters involved in stomata closure. The conserved phenylalanine residue F329 closing the transport pore of SpMae1(p) is essential for the transporter activity. The voltage-dependent SLAC1 transporters do not use proton or Na+ motive force and are, thus, less energetically expensive than the majority of other dicarboxylic acid transporters. Such transporters present a tremendous advantage for organic acid production via fermentation allowing a higher overall product yield.
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Drew, M. E., C. K. Langford, E. M. Klamo, D. G. Russell, M. P. Kavanaugh, and S. M. Landfear. "Functional expression of a myo-inositol/H+ symporter from Leishmania donovani." Molecular and Cellular Biology 15, no. 10 (October 1995): 5508–15. http://dx.doi.org/10.1128/mcb.15.10.5508.
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The vast majority of surface molecules in such kinetoplastid protozoa as members of the genus Leishmania contain inositol and are either glycosyl inositol phospholipids or glycoproteins that are tethered to the external surface of the plasma membrane by glycosylphosphatidylinositol anchors. We have shown that the biosynthetic precursor for these abundant glycolipids, myo-inositol, is translocated across the parasite plasma membrane by a specific transporter that is structurally related to mammalian facilitative glucose transporters. This myo-inositol transporter has been expressed and characterized in Xenopus laevis oocytes. Two-electrode voltage clamp experiments demonstrate that this protein is a sodium-independent electrogenic symporter that appears to utilize a proton gradient to concentrate myo-inositol within the cell. Immunolocalization experiments with a transporter-specific polyclonal antibody reveal the presence of this protein in the parasite plasma membrane.
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Wolken, Wout A. M., Patrick M. Lucas, Aline Lonvaud-Funel, and Juke S. Lolkema. "The Mechanism of the Tyrosine Transporter TyrP Supports a Proton Motive Tyrosine Decarboxylation Pathway in Lactobacillus brevis." Journal of Bacteriology 188, no. 6 (March 15, 2006): 2198–206. http://dx.doi.org/10.1128/jb.188.6.2198-2206.2006.
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ABSTRACT The tyrosine decarboxylase operon of Lactobacillus brevis IOEB9809 contains, adjacent to the tyrosine decarboxylase gene, a gene for TyrP, a putative tyrosine transporter. The two genes potentially form a proton motive tyrosine decarboxylation pathway. The putative tyrosine transporter gene of L. brevis was expressed in Lactococcus lactis and functionally characterized using right-side-out membranes. The transporter very efficiently catalyzes homologous tyrosine-tyrosine exchange and heterologous exchange between tyrosine and its decarboxylation product tyramine. Tyrosine-tyramine exchange was shown to be electrogenic. In addition to the exchange mode, the transporter catalyzes tyrosine uniport but at a much lower rate. Analysis of the substrate specificity of the transporter by use of a set of 19 different tyrosine substrate analogues showed that the main interactions between the protein and the substrates involve the amino group and the phenyl ring with the para hydroxyl group. The carboxylate group that is removed in the decarboxylation reaction does not seem to contribute to the affinity of the protein for the substrates significantly. The properties of the TyrP protein are those typical for precursor-product exchangers that operate in proton motive decarboxylation pathways. It is proposed that tyrosine decarboxylation in L. brevis results in proton motive force generation by an indirect proton pumping mechanism.
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Robinson, Anne E., Nathan E. Thomas, Emma A. Morrison, Bryan M. Balthazor, and Katherine A. Henzler-Wildman. "New free-exchange model of EmrE transport." Proceedings of the National Academy of Sciences 114, no. 47 (November 7, 2017): E10083—E10091. http://dx.doi.org/10.1073/pnas.1708671114.
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EmrE is a small multidrug resistance transporter found in Escherichia coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug. The functional consequence of this finding is an exceptionally promiscuous transporter: not only can EmrE export diverse drug substrates, it can couple antiport of a drug to either one or two protons, performing both electrogenic and electroneutral transport of a single substrate. We present a free-exchange model for EmrE antiport that is consistent with these results and recapitulates ∆pH-driven concentrative drug uptake. Kinetic modeling suggests that free exchange by EmrE sacrifices coupling efficiency but boosts initial transport speed and drug release rate, which may facilitate efficient multidrug efflux.
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Watzke, Natalie, Thomas Rauen, Ernst Bamberg, and Christof Grewer. "On the Mechanism of Proton Transport by the Neuronal Excitatory Amino Acid Carrier 1." Journal of General Physiology 116, no. 5 (October 16, 2000): 609–22. http://dx.doi.org/10.1085/jgp.116.5.609.
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Uptake of glutamate from the synaptic cleft is mediated by high affinity transporters and is driven by Na+, K+, and H+ concentration gradients across the membrane. Here, we characterize the molecular mechanism of the intracellular pH change associated with glutamate transport by combining current recordings from excitatory amino acid carrier 1 (EAAC1)–expressing HEK293 cells with a rapid kinetic technique with a 100-μs time resolution. Under conditions of steady state transport, the affinity of EAAC1 for glutamate in both the forward and reverse modes is strongly dependent on the pH on the cis-side of the membrane, whereas the currents at saturating glutamate concentrations are hardly affected by the pH. Consistent with this, the kinetics of the pre–steady state currents, measured after saturating glutamate concentration jumps, are not a function of the pH. In addition, we determined the deuterium isotope effect on EAAC1 kinetics, which is in agreement with proton cotransport but not OH− countertransport. The results can be quantitatively explained with an ordered binding model that includes a rapid proton binding step to the empty transporter followed by glutamate binding and translocation of the proton-glutamate-transporter complex. The apparent pK of the extracellular proton binding site is ∼8. This value is shifted to ∼6.5 when the substrate binding site is exposed to the cytoplasm.
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Clark, F. S., T. Parkinson, C. A. Hitchcock, and N. A. Gow. "Correlation between rhodamine 123 accumulation and azole sensitivity in Candida species: possible role for drug efflux in drug resistance." Antimicrobial Agents and Chemotherapy 40, no. 2 (February 1996): 419–25. http://dx.doi.org/10.1128/aac.40.2.419.
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A wide variety of prokaryotic and eukaryotic cells exhibit a multidrug resistance (MDR) phenotype, indicating that resistance to potentially toxic compounds is mediated by their active efflux from the cell. We have sought to determine whether resistance to azoles in some strains of Candida species may be due in part to active drug efflux. Rhodamine 123 (Rh123) is a fluorescent compound that is transported by a wide variety of MDR cell types. We have shown that certain azole-resistant strains of Candida albicans, C. glabrata, and C. krusei accumulate less Rh123 than azole-susceptible ones. In C. albicans, Rh123 accumulation was growth phase and temperature dependent and was increased by proton uncouplers and by reserpine, an MDR modulator. This is consistent with an energy-dependent efflux mechanism for Rh123, mediated by an MDR transporter. In C. glabrata, but not in C. albicans, there was competition between Rh123 and fluconazole for efflux. Thus, in C. glabrata, Rh123 and fluconazole appear to be transported via a common MDR-like transporter, whereas in C. albicans, the Rh123 transporter does not appear to transport azoles.
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Casteilla, Louis, Anne Devin, Bénédicte Salin, Nicole Averet, and Michel Rigoulet. "UCP1 as a water/proton co-transporter." Mitochondrion 12, no. 4 (July 2012): 480–81. http://dx.doi.org/10.1016/j.mito.2012.04.003.
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Hirata, Takahiro, Asami Saito, Kunihiko Nishino, Norihisa Tamura, and Akihito Yamaguchi. "Effects of Efflux Transporter Genes on Susceptibility of Escherichia coli to Tigecycline (GAR-936)." Antimicrobial Agents and Chemotherapy 48, no. 6 (June 2004): 2179–84. http://dx.doi.org/10.1128/aac.48.6.2179-2184.2004.
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ABSTRACT The activity of tigecycline, 9-(t-butylglycylamido)-minocycline, against Escherichia coli KAM3 (acrB) strains harboring plasmids encoding various tetracycline-specific efflux transporter genes, tet(B), tet(C), and tet(K), and multidrug transporter genes, acrAB, acrEF, and bcr, was examined. Tigecycline showed potent activity against all three Tet-expressing, tetracycline-resistant strains, with the MICs for the strains being equal to that for the host strain. In the Tet(B)-containing vesicle study, tigecycline did not significantly inhibit tetracycline efflux-coupled proton translocation and at 10 μM did not cause proton translocation. This suggests that tigecycline is not recognized by the Tet efflux transporter at a low concentration; therefore, it exhibits significant antibacterial activity. These properties can explain its potent activity against bacteria with a Tet efflux resistance determinant. Tigecycline induced the Tet(B) protein approximately four times more efficiently than tetracycline, as determined by Western blotting, indicating that it is at least recognized by a TetR repressor. The MICs for multidrug efflux proteins AcrAB and AcrEF were increased fourfold. Tigecycline inhibited active ethidium bromide efflux from intact E. coli cells overproducing AcrAB. Therefore, tigecycline is a possible substrate of AcrAB and its close homolog, AcrEF, which are resistance-modulation-division-type multicomponent efflux transporters.
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Bartoccioni, Paola, Joana Fort, Antonio Zorzano, Ekaitz Errasti-Murugarren, and Manuel Palacín. "Functional characterization of the alanine-serine-cysteine exchanger of Carnobacterium sp AT7." Journal of General Physiology 151, no. 4 (January 29, 2019): 505–17. http://dx.doi.org/10.1085/jgp.201812195.
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Many key cell processes require prior cell uptake of amino acids from the environment, which is facilitated by cell membrane amino acid transporters such as those of the L-type amino acid transporter (LAT) subfamily. Alterations in LAT subfamily amino acid transport are associated with several human diseases, including cancer, aminoacidurias, and neurodegenerative conditions. Therefore, from the perspective of human health, there is considerable interest in obtaining structural information about these transporter proteins. We recently solved the crystal structure of the first LAT transporter, the bacterial alanine-serine-cysteine exchanger of Carnobacterium sp AT7 (BasC). Here, we provide a complete functional characterization of detergent-purified, liposome-reconstituted BasC transporter to allow the extension of the structural insights into mechanistic understanding. BasC is a sodium- and proton-independent small neutral amino acid exchanger whose substrate and inhibitor selectivity are almost identical to those previously described for the human LAT subfamily member Asc-1. Additionally, we show that, like its human counterparts, this transporter has apparent affinity asymmetry for the intra- and extracellular substrate binding sites—a key feature in the physiological role played by these proteins. BasC is an excellent paradigm of human LAT transporters and will contribute to our understanding of the molecular mechanisms underlying substrate recognition and translocation at both sides of the plasma membrane.
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Ferreira, Mário José, and Isabel de Sá-Nogueira. "A Multitask ATPase Serving Different ABC-Type Sugar Importers in Bacillus subtilis." Journal of Bacteriology 192, no. 20 (August 6, 2010): 5312–18. http://dx.doi.org/10.1128/jb.00832-10.
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ABSTRACT Bacillus subtilis is able to utilize arabinopolysaccharides derived from plant biomass. Here, by combining genetic and physiological analyses we characterize the AraNPQ importer and identify primary and secondary transporters of B. subtilis involved in the uptake of arabinosaccharides. We show that the ABC-type importer AraNPQ is involved in the uptake of α-1,5-arabinooligosaccharides, at least up to four l-arabinosyl units. Although this system is the key transporter for α-1,5-arabinotriose and α-1,5-arabinotetraose, the results indicate that α-1,5-arabinobiose also is translocated by the secondary transporter AraE. This broad-specificity proton symporter is the major transporter for arabinose and also is accountable for the uptake of xylose and galactose. In addition, MsmX is shown to be the ATPase that energizes the incomplete AraNPQ importer. Furthermore, the results suggest the existence of at least one more unidentified MsmX-dependent ABC importer responsible for the uptake of nonlinear α-1,2- and α-1,3-arabinooligosaccharides. This study assigns MsmX as a multipurpose B. subtilis ATPase required to energize different saccharide transporters, the arabinooligosaccharide-specific AraNPQ-MsmX system, a putative MsmX-dependent ABC transporter specific for nonlinear arabinooligosaccharides, and the previously characterized maltodextrin-specific MdxEFG-MsmX system.
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Kleinman, J. G. "Proton ATPases and urinary acidification." Journal of the American Society of Nephrology 5, no. 5 (November 1994): S6. http://dx.doi.org/10.1681/asn.v55s6.
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Acidification of the urine is mediated by vectorial H+ transport from cells at a number of sites in the kidney. A proton ATPase has been described that appears to mediate a significant proportion of this H+ transport. In particular, in proximal tubule and collecting duct, there is evidence both for the presence of transporter protein and for H+ transport with features that have been identified with it. This review highlights some of the unresolved questions regarding this transporter, specifically, its distribution and relationship to the vacuolar pump present in endocytotic vesicles, how physiologic control is asserted, and its role in pathophysiology. The review discusses in greater detail the issue of whether the vacuolar H+ ATPase is responsible for all of the urinary acidification and concludes that it probably is not. Specifically, compelling evidence for acidification at sites in the kidney that appear to lack this transporter is presented. In addition, the evidence for the presence in the kidney of a gastric-type H(+)-K+ ATPase is also reviewed. The evidence appears to be strong for a K(+)-stimulated ATPase that is sensitive to omeprazole and SCH 28080, the prototypical H(+)-K+ ATPase inhibitors; however, uncertainties remain because of problems of transport inhibition specificity and discordant results of molecular biologic studies.
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Daniel, Hannelore, and Isabel Rubio-Aliaga. "An update on renal peptide transporters." American Journal of Physiology-Renal Physiology 284, no. 5 (May 1, 2003): F885—F892. http://dx.doi.org/10.1152/ajprenal.00123.2002.
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The brush-border membrane of renal epithelial cells contains PEPT1 and PEPT2 proteins that are rheogenic carriers for short-chain peptides. The carrier proteins display a distinct surface expression pattern along the proximal tubule, suggesting that initially di- and tripeptides, either filtered or released by surface-bound hydrolases from larger oligopeptides, are taken up by the low-affinity but high-capacity PEPT1 transporter and then by PEPT2, which possesses a higher affinity but lower transport capacity. Both carriers transport essentially all possible di- and tripeptides and numerous structurally related drugs. A unique feature of the mammalian peptide transporters is the capability of proton-dependent electrogenic cotransport of all substrates, regardless of their charge, that is achieved by variable coupling in proton movement along with the substrate down the transmembrane potential difference. This review focuses on the postcloning research efforts to understand the molecular physiology of peptide transport processes in renal tubules and summarizes available data on the underlying genes, protein structures, and transporter function as derived from studies in heterologous expression systems.
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Otto, C., S. tom Dieck, and K. Bauer. "Dipeptide uptake by adenohypophysial folliculostellate cells." American Journal of Physiology-Cell Physiology 271, no. 1 (July 1, 1996): C210—C217. http://dx.doi.org/10.1152/ajpcell.1996.271.1.c210.
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Dipeptide uptake was studied in primary cultures from rat anterior pituitaries by use of radiolabeled carnosine and the fluorescent dipeptide derivative beta-Ala-Lys-N epsilon-AMCA (AMCA is 7-amino-4-methylcoumarin-3-acetic acid). Fluorescence microscopic studies revealed that the reporter peptide specifically accumulated in the S-100 positive folliculostellate cells that do not produce any known hormone. The dipeptide derivative was taken up in unmetabolized form by an energy-dependent saturable process with apparent kinetic constants as follows: Michaelis constant, 19 microM; maximum velocity, 5.5 nmol.mg protein-1.h-1. This high-affinity transporter was strongly affected by inhibitors of sodium/proton exchangers and thus appeared to be driven by a proton gradient. Competition studies revealed that the peptide transporter exhibits broad substrate specificity with a preference for hydrophobic dipeptides. In contrast to free amino acids and the pseudotetrapeptide amastatin, tripeptides were also accepted. Compounds without an alpha- and beta-amino group, such as captopril, thiorphan, and benzylpenicillin, did not affect uptake of the reporter peptide, although they were substrates of the well-characterized intestinal and renal dipeptide transporters.
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Mboge, Mam Y., Zhijuan Chen, Daniel Khokhar, Alyssa Wolff, Lingbao Ai, Coy D. Heldermon, Murat Bozdag, et al. "A non-catalytic function of carbonic anhydrase IX contributes to the glycolytic phenotype and pH regulation in human breast cancer cells." Biochemical Journal 476, no. 10 (May 28, 2019): 1497–513. http://dx.doi.org/10.1042/bcj20190177.
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AbstractThe most aggressive and invasive tumor cells often reside in hypoxic microenvironments and rely heavily on rapid anaerobic glycolysis for energy production. This switch from oxidative phosphorylation to glycolysis, along with up-regulation of the glucose transport system, significantly increases the release of lactic acid from cells into the tumor microenvironment. Excess lactate and proton excretion exacerbate extracellular acidification to which cancer cells, but not normal cells, adapt. We have hypothesized that carbonic anhydrases (CAs) play a role in stabilizing both intracellular and extracellular pH to favor cancer progression and metastasis. Here, we show that proton efflux (acidification) using the glycolytic rate assay is dependent on both extracellular pH (pHe) and CA IX expression. Yet, isoform-selective sulfonamide-based inhibitors of CA IX did not alter proton flux, which suggests that the catalytic activity of CA IX is not necessary for this regulation. Other investigators have suggested the CA IX co-operates with the MCT transport family to excrete protons. To test this possibility, we examined the expression patterns of selected ion transporters and show that members of this family are differentially expressed within the molecular subtypes of breast cancer. The most aggressive form of breast cancer, triple-negative breast cancer, appears to co-ordinately express the monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CA IX). This supports a possible mechanism that utilizes the intramolecular H+ shuttle system in CA IX to facilitate proton efflux through MCT4.
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Ullmann, Roland, Roland Gross, Jörg Simon, Gottfried Unden, and Achim Kröger. "Transport of C4-Dicarboxylates inWolinella succinogenes." Journal of Bacteriology 182, no. 20 (October 15, 2000): 5757–64. http://dx.doi.org/10.1128/jb.182.20.5757-5764.2000.
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ABSTRACT C4-dicarboxylate transport is a prerequisite for anaerobic respiration with fumarate in Wolinella succinogenes, since the substrate site of fumarate reductase is oriented towards the cytoplasmic side of the membrane. W. succinogenes was found to transport C4-dicarboxylates (fumarate, succinate, malate, and aspartate) across the cytoplasmic membrane by antiport and uniport mechanisms. The electrogenic uniport resulted in dicarboxylate accumulation driven by anaerobic respiration. The molar ratio of internal to external dicarboxylate concentration was up to 103. The dicarboxylate antiport was either electrogenic or electroneutral. The electroneutral antiport required the presence of internal Na+, whereas the electrogenic antiport also operated in the absence of Na+. In the absence of Na+, no electrochemical proton potential (Δp) was measured across the membrane of cells catalyzing fumarate respiration. This suggests that the proton potential generated by fumarate respiration is dissipated by the concomitant electrogenic dicarboxylate antiport. Three gene loci (dcuA,dcuB, and dctPQM) encoding putative C4-dicarboxylate transporters were identified on the genome of W. succinogenes. The predicted gene products ofdcuA and dcuB are similar to the Dcu transporters that are involved in the fumarate respiration ofEscherichia coli with external C4-dicarboxylates. The genes dctP, -Q, and -M probably encode a binding-protein-dependent secondary uptake transporter for dicarboxylates. A mutant (DcuA− DcuB−) ofW. succinogenes lacking the intact dcuA anddcuB genes grew by nitrate respiration with succinate as the carbon source but did not grow by fumarate respiration with fumarate, malate, or aspartate as substrates. The DcuA−, DcuB−, and DctQM− mutants grew by fumarate respiration as well as by nitrate respiration with succinate as the carbon source. Cells of the DcuA− DcuB−mutant performed fumarate respiration without generating a proton potential even in the presence of Na+. This explains why the DcuA− DcuB− mutant does not grow by fumarate respiration. Growth by fumarate respiration appears to depend on the function of the Na+-dependent, electroneutral dicarboxylate antiport which is catalyzed exclusively by the Dcu transporters. Dicarboxylate transport via the electrogenic uniport is probably catalyzed by the DctPQM transporter and by a fourth, unknown transporter that may also operate as an electrogenic antiporter.
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Lorch, M., I. Lehner, A. Siarheyeva, D. Basting, N. Pfleger, T. Manolikas, and C. Glaubitz. "NMR and fluorescence spectroscopy approaches to secondary and primary active multidrug efflux pumps." Biochemical Society Transactions 33, no. 4 (August 1, 2005): 873–77. http://dx.doi.org/10.1042/bst0330873.
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Multidrug efflux pumps are found in all major transporter families. Along with a lack of three-dimensional structure information, the mechanism of drug recognition, energy coupling with drug translocation and the catalytic cycle are so far not understood. In the present study, we present first data of a fluorescence-based assay to study the pH-gradient-mediated activity of the multidrug antiporter EmrE, by co-reconstitution with the light-driven proton pump bacteriorhodopsin. In addition to biochemical approaches, the emerging technique, solid-state NMR, can be used for the investigation of these transporters. A number of experiments based on MAS (magic angle sample spinning) NMR are available to provide data on protein structure and dynamics, drug binding and protein–lipid interactions. However, these experiments dictate a number of constraints with respect to sample preparation that will be discussed for proteins from the SMR (small multidrug resistance transporter) family. In addition, 2H-NMR is used to probe protein mobility of Lactococcus lactis ABC transporter, LmrA.
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Drummond, Micah J., Christopher S. Fry, Erin L. Glynn, Kyle L. Timmerman, Jared M. Dickinson, Dillon K. Walker, David M. Gundermann, Elena Volpi, and Blake B. Rasmussen. "Skeletal muscle amino acid transporter expression is increased in young and older adults following resistance exercise." Journal of Applied Physiology 111, no. 1 (July 2011): 135–42. http://dx.doi.org/10.1152/japplphysiol.01408.2010.
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Amino acid transporters and mammalian target of rapamycin complex 1 (mTORC1) signaling are important contributors to muscle protein anabolism. Aging is associated with reduced mTORC1 signaling following resistance exercise, but the role of amino acid transporters is unknown. Young ( n = 13; 28 ± 2 yr) and older ( n = 13; 68 ± 2 yr) subjects performed a bout of resistance exercise. Skeletal muscle biopsies ( vastus lateralis) were obtained at basal and 3, 6, and 24 h postexercise and were analyzed for amino acid transporter mRNA and protein expression and regulators of amino acid transporter transcription utilizing real-time PCR and Western blotting. We found that basal amino acid transporter expression was similar in young and older adults ( P > 0.05). Exercise increased L-type amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, sodium-coupled neutral amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, and cationic amino acid transporter 1/SLC7A1 mRNA expression in both young and older adults ( P < 0.05). L-type amino acid transporter 1 and CD98 protein increased only in younger adults ( P < 0.05). eukaryotic initiation factor 2 α-subunit (S52) increased similarly in young and older adults postexercise ( P < 0.05). Ribosomal protein S6 (S240/244) and activating transcription factor 4 nuclear protein expression tended to be higher in the young, while nuclear signal transducer and activator of transcription 3 (STAT3) (Y705) was higher in the older subjects postexercise ( P < 0.05). These results suggest that the rapid upregulation of amino acid transporter expression following resistance exercise may be regulated differently between the age groups, but involves a combination of mTORC1, activating transcription factor 4, eukaryotic initiation factor 2 α-subunit, and STAT3. We propose an increase in amino acid transporter expression may contribute to enhanced amino acid sensitivity following exercise in young and older adults. In older adults, the increased nuclear STAT3 phosphorylation may be indicative of an exercise-induced stress response, perhaps to export amino acids from muscle cells.
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Xia, Li, Karen Engel, Mingyan Zhou, and Joanne Wang. "Membrane localization and pH-dependent transport of a newly cloned organic cation transporter (PMAT) in kidney cells." American Journal of Physiology-Renal Physiology 292, no. 2 (February 2007): F682—F690. http://dx.doi.org/10.1152/ajprenal.00302.2006.
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Plasma membrane monoamine transporter (PMAT) is a novel membrane transporter recently cloned and characterized in our laboratory. We previously demonstrated that PMAT functions as a polyspecific organic cation transporter and efficiently transports many organic cations such as monoamine neurotransmitters and 1-methyl-4-phenylpyridinium (MPP+). In this study, we explored the role of PMAT in the renal handling of organic cations. Using a polyclonal antibody generated toward the NH2-terminal 66 amino acid residues of human PMAT, we showed that the PMAT protein (∼55 kDa) is expressed in the human kidney and is primarily targeted to the apical membranes when expressed in polarized Madin-Darby canine kidney (MDCK) cells. Using MDCK cells stably expressing human PMAT, we showed that PMAT-mediated MPP+ uptake is strongly dependent on extracellular pH. Lowering extracellular pH from 7.4 to 6.6 greatly stimulated PMAT-mediated MPP+ uptake, whereas elevating extracellular pH to 8.2 abolished transporter activity. Kinetic analysis revealed that the apparent Vmax at pH 6.6 is about fourfold higher than that at pH 7.4, whereas the apparent Km values were not statistically different at these two conditions. Under acidic conditions (pH 6.6), the proton ionophore, carbonyl cyanide p-trifluormethoxyphenylhydrazone, drastically reduced PMAT-mediated MPP+ uptake, suggesting that the stimulatory effect of proton may be due to transporter coupling with a proton gradient. Taken together, our data suggest that PMAT is expressed on the apical membranes of renal epithelial cells and may use luminal proton gradient to drive organic cation reabsorption in the kidney.
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Shimoyama, Y., Y. Akihara, D. Kirat, H. Iwano, K. Hirayama, Y. Kagawa, T. Ohmachi, et al. "Expression of Monocarboxylate Transporter 1 in Oral and Ocular Canine Melanocytic Tumors." Veterinary Pathology 44, no. 4 (July 2007): 449–57. http://dx.doi.org/10.1354/vp.44-4-449.
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Solid tumors are composed of a heterogeneous population of cells surviving in various concentrations of oxygen. In a hypoxic environment, tumor cells generally up-regulate glycolysis and, therefore, generate more lactate that must be expelled from the cell through proton transporters to prevent intracellular acidosis. Monocarboxylate transporter 1 (MCT1) is a major proton transporter in mammalian cells that transports monocarboxylates, such as lactate and pyruvate, together with a proton across the plasma membrane. Melanocytic neoplasia occurs frequently in dogs, but the prognosis is highly site-dependent. In this study, 50 oral canine melanomas, which were subdivided into 3 histologic subtypes, and 17 ocular canine melanocytic neoplasms (14 melanocytomas and 3 melanomas) were used to examine and compare MCT1 expression. Immunohistochemistry using a polyclonal chicken anti-rat MCT1 antibody showed that most oral melanoma exhibited cell membrane staining, although there were no significant differences observed among the 3 histologic subtypes. In contrast, the majority of ocular melanocytic tumors were not immunoreactive. Additionally, we documented the presence of a 45-kDa band in cell membrane protein Western blots, and sequencing of a reverse transcriptase polymerase chain reaction band of expected size confirmed its identity as a partial canine MCT1 transcript in 3 oral tumors. Increased MCT1 expression in oral melanomas compared with ocular melanocytic tumors may reflect the very different biology between these tumors in dogs. These results are the first to document canine MCT1 expression in canine tumors and suggest that increased MCT1 expression may provide a potential therapeutic target for oral melanoma.