Abstract
The proton-linked monocarboxylate transporters (MCTs) are required for lactic acid transport into and out of all mammalian cells. Thus, they play an essential role in tumour cells that are usually highly glycolytic and are promising targets for anti-cancer drugs. AR-C155858 is a potent MCT1 inhibitor (Ki ~2 nM) that also inhibits MCT2 when associated with basigin but not MCT4. Previous work [Ovens, M.J. et al. (2010) Biochem. J. 425, 523–530] revealed that AR-C155858 binding to MCT1 occurs from the intracellular side and involves transmembrane helices (TMs) 7–10. In the present paper, we generate a molecular model of MCT4 based on our previous models of MCT1 and identify residues in the intracellular substrate-binding cavity that differ significantly between MCT4 and MCT1/MCT2 and so might account for differences in inhibitor binding. We tested their involvement using site-directed mutagenesis (SDM) of MCT1 to change residues individually or in combination with their MCT4 equivalent and determined inhibitor sensitivity following expression in Xenopus oocytes. Phe360 and Ser364 were identified as important for AR-C155858 binding with the F360Y/S364G mutant exhibiting >100-fold reduction in inhibitor sensitivity. To refine the binding site further, we used molecular dynamics (MD) simulations and additional SDM. This approach implicated six more residues whose involvement was confirmed by both transport studies and [3H]-AR-C155858 binding to oocyte membranes. Taken together, our data imply that Asn147, Arg306 and Ser364 are important for directing AR-C155858 to its final binding site which involves interaction of the inhibitor with Lys38, Asp302 and Phe360 (residues that also play key roles in the translocation cycle) and also Leu274 and Ser278.
Highlights
Almost all mammalian cells require L-lactic acid to be transported across their plasma membranes
Previous work using chimeras of MCT1 and MCT4 indicated that the binding site for AR-C155858 lies in transmembrane helix (TM) 7–10 of the C-terminal domain of MCT1 and is accessed from the inside of the cell [25]
This was created by alignment of the MCT1 and MCT4 sequences followed by building the MCT1 model on the previously established MCT1 model (MCT1_IO), in turn based on the crystal structure of glycerol-3phosphate transporter (GlpT)
Summary
Almost all mammalian cells require L-lactic acid to be transported across their plasma membranes. Characterization of the kinetics of MCT4 reveals that it is especially suited for mediating export of lactic acid produced by glycolysis because, unlike MCT1, it has a very low affinity for pyruvate This ensures that only lactate and not pyruvate is lost from the cell, which is important to ensure the NADH generated by glycolysis can be reoxidized [5,6]. The distribution of MCT2 is less well conserved between species, but it is most often expressed in tissues that require high affinity lactic acid uptake for gluconeogenesis (liver and kidney) or oxidation (neurons) This is consistent with its higher affinity for L-lactate than MCT1 [2,4,7]. MCT3 is less well characterized but its expression is limited to the retinal pigment epithelium and choroid plexus where it acts to mediate lactic acid efflux [4,8]
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