Studies on the DIDS-binding Site of Monocarboxylate Transporter 1 Suggest a Homology Model of the Open Conformation and a Plausible Translocation Cycle

Site-directed mutagenesis of MCT1 was performed on exofacial lysines Lys³⁸, Lys⁴⁵, Lys²⁸², and Lys⁴¹³. 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 SO₃^? groups interact with positively charged Lys²⁸² together with Lys⁴⁵ and/or Lys⁴¹³. This positions one DIDS isothiocyanate group close to Lys³⁸, leading to its covalent modification and irreversible inhibition. Additional mutagenesis revealed that DIDS cross-links MCT1 to its ancillary protein embigin using either Lys³⁸ or Lys²⁹⁰ of MCT1 and Lys¹⁶⁰ or Lys¹⁶⁴ 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 Lys³⁸ in a hydrophobic environment at the bottom of a substrate-binding channel. Our model suggests a translocation cycle in which Lys³⁸ accepts a proton before binding lactate. Both the lactate and proton are then passed through the channel via Asp^302? and Asp³⁰⁶⁺, an ion pair already identified as important for transport and located adjacent to Phe³⁶⁰, 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.Monocarboxylate transporter 1 (MCT1)³ is a member of the monocarboxylate transporter family (SLC16) of which there are 14 known members encoded by both the human and mouse genomes (1). All of the members of this family are thought to have 12 transmembrane alpha helices (TMs) with a large loop between TMs 6 and 7 and the C and N termini facing the cytosol (2, 3). The only members of the MCT family that have been shown to catalyze transport of monocarboxylates such as l-lactate across the plasma membrane are isoforms 1–4 (4–8). This transport is proton-linked and leads to the net uptake or release of lactic acid from cells, which is critical for metabolic pathways such as anaerobic glycolysis, gluconeogenesis, and lactate oxidation (9). MCT8 is a high affinity thyroid hormone transporter (10), whereas MCT10 (TAT1) is an aromatic amino acid transporter (11). The other members of the MCT family remain to be characterized.MCT1 is the most widely distributed member of the MCT family and was first identified as the lactate transporter present in red blood cells where its kinetics and substrate and inhibitor specificity were investigated in detail (9, 11, 12). These studies revealed that MCT1 can be inhibited by stilbene disulfonate derivatives such as DIDS and 4,4′-dibenzamido-stilbene-2,2′-disulfonate (DBDS). DIDS was shown to exhibit a rapid reversible inhibition of transport that was competitive with respect to l-lactate. This is followed by a slowly developing irreversible inhibition that is not exhibited by DBDS and is thought to be caused by one of the isothiocyanate groups of DIDS attacking a lysine residue on MCT1 (13–15). Prolonged incubation with DIDS also led to a fraction of the MCT1 becoming cross-linked to a 70-kDa glycoprotein that was identified as embigin, also known as gp70 (16). Embigin has a short intracellular C terminus, a single TM sequence containing a glutamic acid residue, and a large extracellular N terminus containing two immunoglobulin domains (17, 18). Subsequent studies revealed that either embigin or, more frequently, the homologous protein basigin (also known as CD147) is required as a chaperone to take MCT1 to the membrane (19) where the two proteins must remain associated for transport activity to be maintained (20, 21).Expression of MCTs 1, 2, and 4 in Xenopus laevis oocytes has enabled their further characterization and the effects of site-directed mutagenesis to be investigated (4, 5, 7, 8, 22–24). Such studies, together with homology modeling have enabled us to propose a three-dimensional structure of MCT1 based around the published structure of the Escherichia coli glycerol-3-phosphate transporter (Protein Data Bank 1PW4) (24). This model can account for the effects of mutating a range of amino acids, including some that disrupt the interaction with basigin, and has led to the proposal that the single TM of basigin or embigin lies between TMs 3 and 6 of MCT1. The model also reveals exofacial lysines that are present in MCT1 that might be responsible for the irreversible inhibition of MCT1 by DIDS and the cross-linking of MCT1 to embigin. In rat MCT1 these residues are Lys³⁸, Lys⁴⁵, Lys²⁸², Lys²⁸⁴, Lys²⁹⁰, and Lys⁴¹³. In this paper, we use site-directed mutagenesis of these lysine residues to identify which of them are involved in DIDS binding to MCT1. In addition we use site-directed mutagenesis of embigin to demonstrate that Lys¹⁶⁰ and Lys¹⁶⁴ are involved in its cross-linking to MCT1. Our new data allow us to propose a modified structural model of MCT1 in its outward facing conformation that binds DIDS. This model is consistent with the site-directed mutagenesis data and also suggests a mechanism for the translocation cycle that involves Lys³⁸ as well as Asp³⁰² and Arg³⁰⁶ that have already been identified as important for transport (23, 24). We have also been able to model a structure of MCT1 bound to embigin that is consistent with published data.