Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Biochem J
2010 Jan 15;4253:523-30. doi: 10.1042/BJ20091515.
Show Gene links
Show Anatomy links
AR-C155858 is a potent inhibitor of monocarboxylate transporters MCT1 and MCT2 that binds to an intracellular site involving transmembrane helices 7-10.
Ovens MJ
,
Davies AJ
,
Wilson MC
,
Murray CM
,
Halestrap AP
.
???displayArticle.abstract???
In the present study we characterize the properties of the potent MCT1 (monocarboxylate transporter 1) inhibitor AR-C155858. Inhibitor titrations of L-lactate transport by MCT1 in rat erythrocytes were used to determine the Ki value and number of AR-C155858-binding sites (Et) on MCT1 and the turnover number of the transporter (kcat). Derived values were 2.3+/-1.4 nM, 1.29+/-0.09 nmol per ml of packed cells and 12.2+/-1.1 s-1 respectively. When expressed in Xenopus laevis oocytes, MCT1 and MCT2 were potently inhibited by AR-C155858, whereas MCT4 was not. Inhibition of MCT1 was shown to be time-dependent, and the compound was also active when microinjected, suggesting that AR-C155858 probably enters the cell before binding to an intracellular site on MCT1. Measurement of the inhibitor sensitivity of several chimaeric transporters combining different domains of MCT1 and MCT4 revealed that the binding site for AR-C155858 is contained within the C-terminal half of MCT1, and involves TM (transmembrane) domains 7-10. This is consistent with previous data identifying Phe360 (in TM10) and Asp302 plus Arg306 (TM8) as key residues in substrate binding and translocation by MCT1. Measurement of the Km values of the chimaeras for L-lactate and pyruvate demonstrate that both the C- and N-terminal halves of the molecule influence transport kinetics consistent with our proposed molecular model of MCT1 and its translocation mechanism that requires Lys38 in TM1 in addition to Asp302 and Arg306 in TM8 [Wilson, Meredith, Bunnun, Sessions and Halestrap (2009) J. Biol. Chem. 284, 20011-20021].
Figure 1. Inhibition of L-lactate uptake into rat erythrocytes by AR-C155858Rat erythrocytes were freshly isolated and resuspended at the specified haematocrit as outlined in the Experimental section. Cells were pre-incubated for 1 h at room temperature in the presence or absence of the specified concentration of AR-C155858 (structure shown). Transport was measured by continuous monitoring of the extracellular pH, and calibration of pH changes in terms of proton uptake was achieved by addition of standardized NaOH (10 μM final) just before addition of 10 mM L-lactate to initiate transport. Initial rates of transport were calculated by first-order regression analysis of the time course of pH change. The data were then fitted by non-linear least squares inhibition to the equation for a tight-binding non-competitive inhibitor using inhibitor concentration and haematocrit as the two x-variables [41,42]. The derived values (±S.E. of the fit shown) for the Ki and concentration of binding sites were 2.3±1.4 nM and 1.29±0.09 nmol per ml of packed cells respectively.
Figure 2. MCT1 expressed in Xenopus oocytes is inhibited by AR-C155858 in a time- and concentration-dependent mannerXenopus oocytes were injected with the appropriate cRNA and after 72 h expression were pre-incubated in pH 6 oocyte transport buffer in the presence or absence of 0.1 μM or 1 μM AR-C155858 for the times shown. The uptake of 0.5 mM L-[14C]lactate uptake was then determined after 2.5 min over which period it was found to be linear with time. Data are shown as means±S.E.M of ten separate oocytes. The inset shows the uptake of 20 mM L-lactate measured at pH 6.0 using the pH-sensitive dye BCECF. Data are presented for the same oocyte in the absence of inhibitor and then after 20 min superfusion with 0.1 μM and 1 μM AR-C155858.
Figure 3. When expressed in Xenopus oocytes, MCT1 and MCT2, but not MCT4, are sensitive to inhibition by AR-C155858Xenopus oocytes were injected with appropriate cRNA or water and after 72 h expression they were incubated with the concentration of AR-C155858 shown for 45 min prior to measurement of L-[14C]lactate uptake over 2.5 min. Data are shown as the means±S.E.M. of 15–40 separate oocytes for each inhibitor concentration. The inset images show the expression of the relevant MCT in oocyte sections revealed using immunoflourescence microscopy with C-terminal antibodies against the relevant MCT. Arrows indicate the location of the plasma membrane. Note that for MCT2 a significant proportion of the MCT remains in an intracellular compartment.
Figure 4. Microinjection of AR-C155858 inhibits MCT1 expressed in Xenopus oocytesMCT1 was expressed in Xenopus oocytes for 72 h prior to inhibitor treatment and assay of L-[14C]lactate uptake over 2.5 min. For addition of AR-C155858 internally, 20 oocytes were individually injected with 9.2 nl of 1 mM AR-C155858 or DMSO as a control, incubated for 5 min in 5 ml of pH 6 transport buffer and washed once prior to transport assay. For incubation with the equivalent amount of AR-C155858 added externally, 20 aliquots (9.2 nl) of 1 mM AR-C155858 were added to 5 ml of pH 6 transport buffer (final concentration of 35 nM) and incubated with the oocytes for 5 min prior to a single wash and transport assay as above. Uptake was corrected for the uptake by water-injected eggs under the same conditions and are presented as means±S.E.M. of 18–20 separate oocytes. The two images on the right-hand side show the plasma membrane expression of MCT1 in control oocytes and those incubated for 1 h with 1 μM AR-C155858 revealed by immunofluorescence microscopy.
Figure 5. The binding site of MCT1 for AR-C155858 resides within the C-terminal half of the transporter(A) Shows a schematic of the MCT1/4 and MCT4/1 chimaeras used, with MCT1 sequence being shown in black and MCT4 sequence in grey. The numbered arrows indicate the position that the switch was made between MCT1 and MCT4. Native and chimaeric MCTs were expressed in oocytes for 72 h prior to incubation with AR-C155858 after which assay of L-[14C]lactate uptake was determined as described in Figures 2 and 3. Note that different times of uptake were employed depending on the activity of the chimaera as detailed in Supplementary Table S2 (at http://www.BiochemJ.org/bj/425/bj4250523add.htm). (B) Data on the absolute rate of transport of each chimaera in the absence of inhibitor. (C) The activity at each inhibitor concentration expressed as a percentage of the uninhibited rate, after the background uptake rate of water-injected oocytes was subtracted. Data are shown as the means±S.E.M. of 20–50 separate oocytes for each condition. The inset images of (B) show the plasma membrane expression of each native and chimaeric MCT revealed by immunofluorescence microscopy.
Figure 6. The C-terminus of MCT1 is not involved in AR-C155858 sensitivity(A) Shows a schematic of the MCT1/4 and MCT4/1 chimaeras used, with MCT1 sequence being shown in black and MCT4 sequence in grey. The numbered arrows indicate the position that the switch was made between MCT1 and MCT4. Native and chimaeric MCTs were expressed in oocytes for 72 h prior to incubation with or without 0.1 μM AR-C155858 for 45 min, after which assay of L-[14C]lactate uptake was determined over 2.5 min as described in Figures 2 and 3. (B) Provides mean data (±S.E.M. for ten oocytes) on the absolute rate of transport of each chimaera in the absence and presence of inhibitor. The inset images show the plasma membrane expression of each native and chimaeric MCT revealed by immunofluorescence microscopy.
Figure 7. A region within TMs 7–10 of MCT1 is required for sensitivity to AR-C155858(A) Shows a schematic of the MCT1/4 and MCT4/1 chimaeras used, with MCT1 sequence being shown in black and MCT4 sequence in grey. The numbered arrows indicate the position that the switch was made between MCT1 and MCT4. Native and chimaeric MCTs were expressed in oocytes for 72 h prior to incubation with or without 0.1 μM AR-C155858 for 45 min, after which assay of L-[14C]lactate uptake was determined over 1 h. This prolonged uptake period was required because of the low rates of transport being measured. (B) Provides mean data (±S.E.M. for ten oocytes) on the absolute rate of transport of each chimaera in the absence and presence of inhibitor. The inset images show the plasma membrane expression of each native and chimaeric MCT revealed by immunofluorescence microscopy.
Bergersen,
Is lactate food for neurons? Comparison of monocarboxylate transporter subtypes in brain and muscle.
2007, Pubmed
Bergersen,
Is lactate food for neurons? Comparison of monocarboxylate transporter subtypes in brain and muscle.
2007,
Pubmed
Bröer,
Characterization of the monocarboxylate transporter 1 expressed in Xenopus laevis oocytes by changes in cytosolic pH.
1998,
Pubmed
,
Xenbase
Bröer,
Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes.
1999,
Pubmed
,
Xenbase
Carpenter,
The kinetics, substrate and inhibitor specificity of the lactate transporter of Ehrlich-Lettre tumour cells studied with the intracellular pH indicator BCECF.
1994,
Pubmed
Carruthers,
Facilitated diffusion of glucose.
1990,
Pubmed
Choi,
Role of extracellular domains in PBAN/pyrokinin GPCRs from insects using chimera receptors.
2007,
Pubmed
Davidson,
Partial inhibition by cyclosporin A of the swelling of liver mitochondria in vivo and in vitro induced by sub-micromolar [Ca2+], but not by butyrate. Evidence for two distinct swelling mechanisms.
1990,
Pubmed
Deuticke,
Discrimination of three parallel pathways of lactate transport in the human erythrocyte membrane by inhibitors and kinetic properties.
1982,
Pubmed
Dimmer,
The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells.
2000,
Pubmed
,
Xenbase
Ekberg,
The specific monocarboxylate transporter-1 (MCT-1) inhibitor, AR-C117977, induces donor-specific suppression, reducing acute and chronic allograft rejection in the rat.
2007,
Pubmed
Erlichman,
Inhibition of monocarboxylate transporter 2 in the retrotrapezoid nucleus in rats: a test of the astrocyte-neuron lactate-shuttle hypothesis.
2008,
Pubmed
Fang,
The H+-linked monocarboxylate transporter (MCT1/SLC16A1): a potential therapeutic target for high-risk neuroblastoma.
2006,
Pubmed
Friesema,
Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter.
2003,
Pubmed
,
Xenbase
Garcia,
Molecular characterization of a membrane transporter for lactate, pyruvate, and other monocarboxylates: implications for the Cori cycle.
1994,
Pubmed
Garcia,
cDNA cloning of MCT2, a second monocarboxylate transporter expressed in different cells than MCT1.
1995,
Pubmed
Grollman,
Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast.
2000,
Pubmed
Guile,
Potent blockers of the monocarboxylate transporter MCT1: novel immunomodulatory compounds.
2006,
Pubmed
Guile,
Optimization of monocarboxylate transporter 1 blockers through analysis and modulation of atropisomer interconversion properties.
2007,
Pubmed
Halestrap,
The SLC16 gene family-from monocarboxylate transporters (MCTs) to aromatic amino acid transporters and beyond.
2004,
Pubmed
Halestrap,
Specific inhibition of pyruvate transport in rat liver mitochondria and human erythrocytes by alpha-cyano-4-hydroxycinnamate.
1974,
Pubmed
Halestrap,
The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors.
1975,
Pubmed
Halestrap,
Transport of pyruvate nad lactate into human erythrocytes. Evidence for the involvement of the chloride carrier and a chloride-independent carrier.
1976,
Pubmed
Halestrap,
Lactate transport in heart in relation to myocardial ischemia.
1997,
Pubmed
Halestrap,
The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation.
1999,
Pubmed
,
Xenbase
Helgerson,
Analysis of protein-mediated 3-O-methylglucose transport in rat erythrocytes: rejection of the alternating conformation carrier model for sugar transport.
1989,
Pubmed
Jackson,
Cloning of the monocarboxylate transporter isoform MCT2 from rat testis provides evidence that expression in tissues is species-specific and may involve post-transcriptional regulation.
1997,
Pubmed
Juel,
Lactate transport in skeletal muscle - role and regulation of the monocarboxylate transporter.
1999,
Pubmed
Kim,
cDNA cloning of MEV, a mutant protein that facilitates cellular uptake of mevalonate, and identification of the point mutation responsible for its gain of function.
1992,
Pubmed
Kirk,
CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression.
2000,
Pubmed
Manning Fox,
Characterisation of human monocarboxylate transporter 4 substantiates its role in lactic acid efflux from skeletal muscle.
2000,
Pubmed
,
Xenbase
Manoharan,
The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity.
2006,
Pubmed
,
Xenbase
McCullagh,
Role of the lactate transporter (MCT1) in skeletal muscles.
1996,
Pubmed
Meredith,
The SLC16 monocaboxylate transporter family.
2008,
Pubmed
Murray,
Monocarboxylate transporter MCT1 is a target for immunosuppression.
2005,
Pubmed
Nishimura,
Kinetics of GLUT1 and GLUT4 glucose transporters expressed in Xenopus oocytes.
1993,
Pubmed
,
Xenbase
Philp,
Mouse MCT3 gene is expressed preferentially in retinal pigment and choroid plexus epithelia.
2001,
Pubmed
Pierre,
Monocarboxylate transporters in the central nervous system: distribution, regulation and function.
2005,
Pubmed
Poole,
Reversible and irreversible inhibition, by stilbenedisulphonates, of lactate transport into rat erythrocytes. Identification of some new high-affinity inhibitors.
1991,
Pubmed
Poole,
Transport of lactate and other monocarboxylates across mammalian plasma membranes.
1993,
Pubmed
Poole,
N-terminal protein sequence analysis of the rabbit erythrocyte lactate transporter suggests identity with the cloned monocarboxylate transport protein MCT1.
1994,
Pubmed
Poole,
Studies of the membrane topology of the rat erythrocyte H+/lactate cotransporter (MCT1).
1996,
Pubmed
Rae,
Metabolic effects of blocking lactate transport in brain cortical tissue slices using an inhibitor specific to MCT1 and MCT2.
2009,
Pubmed
Shearman,
The concentration of the mitochondrial pyruvate carrier in rat liver and heart mitochondria determined with alpha-cyano-beta-(1-phenylindol-3-yl)acrylate.
1984,
Pubmed
Sonveaux,
Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice.
2008,
Pubmed
Stegen,
Swelling-induced taurine release without chloride channel activity in Xenopus laevis oocytes expressing anion channels and transporters.
2000,
Pubmed
,
Xenbase
Ullah,
The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1alpha-dependent mechanism.
2006,
Pubmed
Wilson,
Studies on the DIDS-binding site of monocarboxylate transporter 1 suggest a homology model of the open conformation and a plausible translocation cycle.
2009,
Pubmed
,
Xenbase
Wilson,
Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3.
1998,
Pubmed
Wilson,
Basigin (CD147) is the target for organomercurial inhibition of monocarboxylate transporter isoforms 1 and 4: the ancillary protein for the insensitive MCT2 is EMBIGIN (gp70).
2005,
Pubmed
,
Xenbase
Yoon,
Identification of a unique monocarboxylate transporter (MCT3) in retinal pigment epithelium.
1997,
Pubmed