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J Biol Chem. July 24, 2009; 284 (30): 20011-21.

Studies on the DIDS-binding site of monocarboxylate transporter 1 suggest a homology model of the open conformation and a plausible translocation cycle.

Wilson MC , Meredith D , Bunnun C , Sessions RB , Halestrap AP .

Site-directed mutagenesis of MCT1 was performed on exofacial lysines Lys(38), Lys(45), Lys(282), and Lys(413). K38Q-MCT1 and K38R-MCT1 were inactive when expressed at the plasma membrane of Xenopus laevis oocytes, whereas K45R/K282R/K413R-MCT1 and K45Q/K282Q/K413Q-MCT1 were active. The former exhibited normal reversible and irreversible inhibition by DIDS, whereas the latter showed less reversible and no irreversible inhibition. K45Q/K413Q-MCT1 retained some irreversible inhibition, whereas K45Q/K282Q-MCT1 and K282Q/K413Q-MCT1 did not. These data suggest that the two DIDS SO(3)(-) groups interact with positively charged Lys(282) together with Lys(45) and/or Lys(413). This positions one DIDS isothiocyanate group close to Lys(38), leading to its covalent modification and irreversible inhibition. Additional mutagenesis revealed that DIDS cross-links MCT1 to its ancillary protein embigin using either Lys(38) or Lys(290) of MCT1 and Lys(160) or Lys(164) of embigin. We have modeled a possible structure for the outward facing (open) conformation of MCT1 by employing modest rotations of the C-terminal domain of the inner facing conformation modeled previously. The resulting model structure has a DIDS-binding site consistent with experimental data and locates Lys(38) in a hydrophobic environment at the bottom of a substrate-binding channel. Our model suggests a translocation cycle in which Lys(38) accepts a proton before binding lactate. Both the lactate and proton are then passed through the channel via Asp(302-) and Asp(306+), an ion pair already identified as important for transport and located adjacent to Phe(360), which controls channel selectivity. The cross-linking data have also been used to model a structure of MCT1 bound to embigin that is consistent with published data.

PubMed ID: 19473976
PMC ID: PMC2740427
Article link: J Biol Chem.
Grant support: 079792 Wellcome Trust , 079792/z/06 Wellcome Trust

Genes referenced: mcts1 prss1 slc16a1

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