Conformationally Sensitive Residues in Extracellular Loop 5 of the Na+/Dicarboxylate Co-transporter

Abstract

The Na+/dicarboxylate co-transporter, NaDC-1, from the kidney and small intestine, transports three sodium ions together with one divalent anion substrate, such as succinate2-. A previous study (Pajor, A. M. (2001) J. Biol. Chem. 276, 29961-29968), identified four amino acids, Ser-478, Ala-480, Ala-481, and Thr-482, near the extracellular end of transmembrane helix (TM) 9 that are likely to form part of the permeation pathway of the transporter. All four cysteine-substituted mutants were sensitive to inhibition by the membrane-impermeant reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate (MTSET) and protected by substrate. In the present study, we continued the cysteine scan through extracellular loop 5 and TM10, from Thr-483 to Val-528. Most cysteine substitutions were well tolerated, although cysteine mutations of some residues, particularly within the TM, produced proteins that were not expressed on the plasma membrane. Six residues in the extracellular loop (Thr-483, Thr-484, Leu-485, Leu-487, Ile-489, and Met-493) were sensitive to chemical labeling by MTSET, depending on the conformational state of the protein. Transport inhibition by MTSET could be prevented by substrate regardless of temperature, suggesting that the likely mechanism of substrate protection is steric hindrance rather than large-scale conformational changes associated with translocation. We conclude that extracellular loop 5 in NaDC-1 appears to have a functional role, and it is likely to be located in or near the substrate translocation pore in the protein. Conformational changes in the protein affect the accessibility of the residues in extracellular loop 5 and provide further evidence of large-scale changes in the structure of NaDC-1 during the transport cycle.