scholarly journals The loop between helix 4 and helix 5 in the monocarboxylate transporter MCT1 is important for substrate selection and protein stability

2003 ◽  
Vol 376 (2) ◽  
pp. 413-422 ◽  
Author(s):  
Sandra GALIĆ ◽  
Hans-Peter SCHNEIDER ◽  
Angelika BRÖER ◽  
Joachim W. DEITMER ◽  
Stefan BRÖER
Keyword(s):  
Mammalian Cells ◽  
Ketone Bodies ◽  
Monocarboxylate Transporter ◽  
Substrate Selection ◽  
Transport Activity ◽  
Complete Inactivation ◽  
Transporter Family ◽  
Monocarboxylate Transport ◽  
Critical Residues ◽  
Signature Sequence

Transport of lactate, pyruvate and the ketone bodies acetoacetate and β-hydroxybutyrate, is mediated in most mammalian cells by members of the monocarboxylate transporter family (SLC16). A conserved signature sequence has been identified in this family, which is located in the loop between helix 4 and helix 5 and extends into helix 5. We have mutated residues in this signature sequence in the rat monocarboxylate transporter (MCT1) to elucidate the significance of this region for monocarboxylate transport. Mutation of R143 and G153 resulted in complete inactivation of the transporter. For the MCT1(G153V) mutant this was explained by a failure to reach the plasma membrane. The lack of transport activity of MCT1(R143Q) could be partially rescued by the conservative exchange R143H. The resulting mutant transporter displayed reduced stability, a decreased Vmax of lactate transport but not of acetate transport, and an increased stereoselectivity. Mutation of K137, K141 and K142 indicated that only K142 played a significant role in the transport mechanism. Mutation of K142 to glutamine resulted in an increase of the Km for lactate from 5 mM to 12 mM. In contrast with MCT1(R143H), MCT1(K142Q) was less stereoselective than the wild-type. A mechanism is proposed that includes all critical residues.

scholarly journals Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes

1999 ◽  
Vol 341 (3) ◽  
pp. 529-535 ◽  
Author(s):  
Stefan BRÖER ◽  
Angelika BRÖER ◽  
Hans-Peter SCHNEIDER ◽  
Carola STEGEN ◽  
Andrew P. HALESTRAP ◽  
...  
Keyword(s):  
Ketone Bodies ◽  
Anion Channel ◽  
Monocarboxylate Transporter ◽  
Xenopus Laevis Oocytes ◽  
Lactate Transport ◽  
High Affinity ◽  
Monocarboxylate Transport ◽  
Km Value ◽  
Decreasing Ph

Observations on lactate transport in brain cells and cardiac myocytes indicate the presence of a high-affinity monocarboxylate transporter. The rat monocarboxylate transporter isoform MCT2 was analysed by expression in Xenopus laevisoocytes and the results were compared with the known characteristics of lactate transport in heart and brain. Monocarboxylate transport via MCT2 was driven by the H+ gradient over the plasma membrane. Uptake of lactate strongly increased with decreasing pH, showing half-maximal stimulation at pH 7.2. A wide variety of monocarboxylates and ketone bodies, including lactate, pyruvate, β-hydroxybutyrate, acetoacetate, 2-oxoisovalerate and 2-oxoisohexanoate, were substrates of MCT2. All substrates had a high affinity for MCT2. For lactate a Km value of 0.74±0.07 mM was determined at pH 7.0. For the other substrates, Ki values between 100 μM and 1 mM were measured for inhibition of lactate transport, which is about one-tenth of the corresponding values for the ubiquitously expressed monocarboxylate transporter isoform MCT1. Monocarboxylate transport via MCT2 could be inhibited by α-cyano-4-hydroxycinnamate, anion-channel inhibitors and flavonoids. It is suggested that cells which express MCT2 preferentially use lactate and ketone bodies as energy sources.

scholarly journals The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells

2000 ◽  
Vol 350 (1) ◽  
pp. 219-227 ◽  
Author(s):  
Kai-Stefan DIMMER ◽  
Björn FRIEDRICH ◽  
Florian LANG ◽  
Joachim W. DEITMER ◽  
Stefan BRÖER
Keyword(s):  
Mammalian Cells ◽  
Short Chain Fatty Acids ◽  
Monocarboxylate Transporter ◽  
Xenopus Laevis Oocytes ◽  
Cytosolic Ph ◽  
Monocarboxylate Transport ◽  
Thiol Reagent ◽  
Km Value ◽  
Flux Measurements ◽  
Disulphonic Acid

Transport of lactate and other monocarboxylates in mammalian cells is mediated by a family of transporters, designated monocarboxylate transporters (MCTs). The MCT4 member of this family has recently been identified as the major isoform of white muscle cells, mediating lactate efflux out of glycolytically active myocytes [Wilson, Jackson, Heddle, Price, Pilegaard, Juel, Bonen, Montgomery, Hutter and Halestrap (1998) J. Biol. Chem. 273, 15920–15926]. To analyse the functional properties of this transporter, rat MCT4 was expressed in Xenopus laevis oocytes and transport activity was monitored by flux measurements with radioactive tracers and by changes of the cytosolic pH using pH-sensitive microelectrodes. Similar to other members of this family, monocarboxylate transport via MCT4 is accompanied by the transport of H+ across the plasma membrane. Uptake of lactate strongly increased with decreasing extracellular pH, which resulted from a concomitant drop in the Km value. MCT4 could be distinguished from the other isoforms mainly in two respects. First, MCT4 is a low-affinity MCT: for l-lactate Km values of 17±3mM (pH-electrode) and 34±5mM (flux measurements with l-[U-14C]lactate) were determined. Secondly, lactate is the preferred substrate of MCT4. Km values of other monocarboxylates were either similar to the Km value for lactate (pyruvate, 2-oxoisohexanoate, 2-oxoisopentanoate, acetoacetate) or displayed much lower affinity for the transporter (β-hydroxybutyrate and short-chain fatty acids). Under physiological conditions, rat MCT will therefore preferentially transport lactate. Monocarboxylate transport via MCT4 could be competitively inhibited by α-cyano-4-hydroxycinnamate, phloretin and partly by 4,4´-di-isothiocyanostilbene-2,2´-disulphonic acid. Similar to MCT1, monocarboxylate transport via MCT4 was sensitive to inhibition by the thiol reagent p-chloromercuribenzoesulphonic acid.

scholarly journals The inhibition of monocarboxylate transporter 2 (MCT2) by AR-C155858 is modulated by the associated ancillary protein

2010 ◽  
Vol 431 (2) ◽  
pp. 217-225 ◽  
Author(s):  
Matthew J. Ovens ◽  
Christine Manoharan ◽  
Marieangela C. Wilson ◽  
Clarey M. Murray ◽  
Andrew P. Halestrap
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Monocarboxylate Transporter ◽  
Xenopus Laevis Oocytes ◽  
Transport Activity ◽  
Membrane Expression ◽  
Binding Partner ◽  
Reverse Transcription Pcr ◽  
C Terminus ◽  
Plasma Membrane Expression

In mammalian cells, MCTs (monocarboxylate transporters) require association with an ancillary protein to enable plasma membrane expression of the active transporter. Basigin is the preferred binding partner for MCT1, MCT3 and MCT4, and embigin for MCT2. In rat and rabbit erythrocytes, MCT1 is associated with embigin and basigin respectively, but its sensitivity to inhibition by AR-C155858 was found to be identical. Using RT (reverse transcription)–PCR, we have shown that Xenopus laevis oocytes contain endogenous basigin, but not embigin. Co-expression of exogenous embigin was without effect on either the expression of MCT1 or its inhibition by AR-C155858. In contrast, expression of active MCT2 at the plasma membrane of oocytes was significantly enhanced by co-expression of exogenous embigin. This additional transport activity was insensitive to inhibition by AR-C155858 unlike that by MCT2 expressed with endogenous basigin that was potently inhibited by AR-C155858. Chimaeras and C-terminal truncations of MCT1 and MCT2 were also expressed in oocytes in the presence and absence of exogenous embigin. L-Lactate Km values for these constructs were determined and revealed that the TM (transmembrane) domains of an MCT, most probably TM7–TM12, but not the C-terminus, are the major determinants of L-lactate affinity, whereas the associated ancillary protein has little or no effect. Inhibitor titrations of lactate transport by these constructs indicated that embigin modulates MCT2 sensitivity to AR-C155858 through interactions with both the intracellular C-terminus and TMs 3 and 6 of MCT2. The C-terminus of MCT2 was found to be essential for its expression with endogenous basigin.

scholarly journals Monocarboxylate transporter expression in mouse brain

1998 ◽  
Vol 275 (3) ◽  
pp. E516-E524 ◽  
Author(s):  
E. M. Koehler-Stec ◽  
I. A. Simpson ◽  
S. J. Vannucci ◽  
K. T. Landschulz ◽  
W. H. Landschulz
Keyword(s):  
Mouse Brain ◽  
Mammalian Cells ◽  
Alternative Energy ◽  
Ketone Bodies ◽  
Cellular Level ◽  
Monocarboxylate Transporter ◽  
Normal Brain ◽  
Monocarboxylic Acids ◽  
Normal Brain Function

Although glucose is the major metabolic fuel needed for normal brain function, monocarboxylic acids, i.e., lactate, pyruvate, and ketone bodies, can also be utilized by the brain as alternative energy substrates. In most mammalian cells, these substrates are transported either into or out of the cell by a family of monocarboxylate transporters (MCTs), first cloned and sequenced in the hamster. We have recently cloned two MCT isoforms (MCT1 and MCT2) from a mouse kidney cDNA library. Northern blot analysis revealed that MCT1 mRNA is ubiquitous and can be detected in most tissues at a relatively constant level. MCT2 expression is more limited, with high levels of expression confined to testes, kidney, stomach, and liver and lower levels in lung, brain, and epididymal fat. Both MCT1 mRNA and MCT2 mRNA are detected in mouse brain using antisense riboprobes and in situ hybridization. MCT1 mRNA is found throughout the cortex, with higher levels of hybridization in hippocampus and cerebellum. MCT2 mRNA was detected in the same areas, but the pattern of expression was more specific. In addition, MCT1 mRNA, but not MCT2, is localized to the choroid plexus, ependyma, microvessels, and white matter structures such as the corpus callosum. These results suggest a differential expression of the two MCTs at the cellular level.

Transport of lactate and other monocarboxylates across mammalian plasma membranes

1993 ◽  
Vol 264 (4) ◽  
pp. C761-C782 ◽  
Author(s):  
R. C. Poole ◽  
A. P. Halestrap
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Ketone Bodies ◽  
Monocarboxylate Transporter ◽  
Transport Systems ◽  
Monocarboxylate Transporters ◽  
Wide Range ◽  
Almost All ◽  
Beta Hydroxybutyrate

Transport of L-lactate across the plasma membrane is of considerable importance to almost all mammalian cells. In most cells a specific H(+)-monocarboxylate cotransporter is largely responsible for this process; the capacity of this carrier is usually very high, to support the high rates of production or utilization of L-lactate. The best characterized H(+)-monocarboxylate transporter is that of the erythrocyte membrane, which transports L-lactate and a wide range of other aliphatic monocarboxylates, including pyruvate and the ketone bodies acetoacetate and beta-hydroxybutyrate. This carrier is inhibited by alpha-cyanocinnamate derivatives and some stilbene disulfonates and has been identified as a protein of 35-50 kDa on the basis of purification and specific labeling experiments. Other cells possess similar alpha-cyanocinnamate-sensitive H(+)-linked monocarboxylate transporters, but in some cases there are significant differences in the properties of these systems, sufficient to suggest the existence of a family of such carriers. In particular, cardiac muscle and tumor cells have transporters that differ in their Km values for certain substrates (including stereoselectivity for L- over D-lactate) and in their sensitivity to inhibitors. Mitochondria, bacteria, and yeast also possess H(+)-monocarboxylate transporters that share some properties in common with those in the mammalian plasma membrane but are adapted to their specific roles. However, there are distinct Na(+)-monocarboxylate cotransporters on the luminal surface of intestinal and kidney epithelia, which enable active uptake of lactate, pyruvate, and ketone bodies in these tissues. This article reviews the properties of these transport systems and their role in mammalian metabolism.

scholarly journals Mechanism(s) of butyrate transport in Caco-2 cells: role of monocarboxylate transporter 1

2000 ◽  
Vol 279 (4) ◽  
pp. G775-G780 ◽  
Author(s):  
Christos Hadjiagapiou ◽  
Larry Schmidt ◽  
Pradeep K. Dudeja ◽  
Thomas J. Layden ◽  
Krishnamurthy Ramaswamy
Keyword(s):  
Short Chain Fatty Acid ◽  
Rnase Protection Assay ◽  
Significant Inhibition ◽  
Monocarboxylate Transporter ◽  
Rnase Protection ◽  
Monocarboxylate Transporter 1 ◽  
Antisense Orientation ◽  
Transporter Family ◽  
Saturation Kinetics

The short-chain fatty acid butyrate was readily taken up by Caco-2 cells. Transport exhibited saturation kinetics, was enhanced by low extracellular pH, and was Na+independent. Butyrate uptake was unaffected by DIDS; however, α-cyano-4-hydroxycinnamate and the butyrate analogs propionate and l-lactate significantly inhibited uptake. These results suggest that butyrate transport by Caco-2 cells is mediated by a transporter belonging to the monocarboxylate transporter family. We identified five isoforms of this transporter, MCT1, MCT3, MCT4, MCT5, and MCT6, in Caco-2 cells by PCR, and MCT1 was found to be the most abundant isoform by RNase protection assay. Transient transfection of MCT1, in the antisense orientation, resulted in significant inhibition of butyrate uptake. The cells fully recovered from this inhibition by 5 days after transfection. In conclusion, our data showed that the MCT1 transporter may play a major role in the transport of butyrate into Caco-2 cells.

Na(+)-independent transport (uniport) of amino acids and glucose in mammalian cells.

1994 ◽  
Vol 196 (1) ◽  
pp. 93-108
Author(s):  
D K Kakuda ◽  
C L MacLeod
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Glucose Transporter ◽  
Sequence Similarity ◽  
Amino Acid Sequence Similarity ◽  
Amino Acid Transporters ◽  
Cationic Amino Acid ◽  
Transporter Family ◽  
Share Amino Acid Sequence

Recent advances have made possible the isolation of the genes and their cDNAs encoding Na(+)-independent amino acid transporters. Two classes of amino acid 'uniporters' have been isolated. One class contains the mCAT (murine cationic amino acid transporter) gene family that encodes proteins predicted to span the membrane 12-14 times and exhibits structural properties similar to the GLUT (glucose transporter) family and to other well-known transporters. The other class consists of two known genes, rBAT (related to B system amino acid transporters) and 4F2hc, that share amino acid sequence similarity with alpha-amylases and alpha-glucosidases. They are type II glycoproteins predicted to span the membrane only once, yet they mediate the Na(+)-independent transport of cationic and zwitterionic amino acids in Xenopus oocytes. Mutations in the human rBAT gene have been identified by Palacín and his co-workers in several families suffering from a heritable form of cystinuria. This important finding clearly establishes a key role for rBAT in cystine transport. The two classes of amino acid transporters are compared with the well-studied GLUT family of Na(+)-independent glucose transporters.

scholarly journals Expression of Monocarboxylate Transporter 1 in Oral and Ocular Canine Melanocytic Tumors

2007 ◽  
Vol 44 (4) ◽  
pp. 449-457 ◽  
Author(s):  
Y. Shimoyama ◽  
Y. Akihara ◽  
D. Kirat ◽  
H. Iwano ◽  
K. Hirayama ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Mammalian Cells ◽  
Monocarboxylate Transporter ◽  
Intracellular Acidosis ◽  
Western Blots ◽  
Monocarboxylate Transporter 1 ◽  
Melanocytic Tumors ◽  
Oral Melanoma ◽  
Cell Membrane Protein ◽  
Histologic Subtypes

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.

Sign in / Sign up

Export Citation Format

Share Document