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

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 V_max 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 K_m 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.