Conformationally sensitive residues in transmembrane domain 9 of the Na+/dicarboxylate co-transporter

The Na+/dicarboxylate co-transporter, NaDC-1, couples the transport of sodi um and Krebs cycle intermediates, such as succinate and citrate. Previous s tudies identified two functionally important amino acids, Glu-475 and Cys-4 76, located in transmembrane domain (TMD) 9 of NaDC-1. In the present study , each amino acid in TMD-9 was mutated to cysteine, one at a time, and the accessibility of the membrane-impermeant reagent [2-(trimethylammonium)ethy l]methanethiosulfonate (MTSET) to the replacement cysteines was determined. Cysteine substitution was tolerated at all but five of the sites: the A461 C mutant was not present at the plasma membrane, whereas the F473C, T474C, E475C, and N479C mutants were inactive proteins located on the plasma membr ane. Cysteine substitution of four residues found near the extracellular su rface of TMD-9 (Ser-478, Ala-480, Ala-481, and Thr-482) resulted in protein s that were sensitive to inhibition by MTSET. The accessibility of MTSET to the four substituted cysteines was highest in the presence of the transpor ted cations, sodium or lithium, and low in choline. The four mutants also e xhibited substrate protection of MTSET accessibility. The MTSET accessibili ty to S478C, A481C, and A480C was independent of voltage. In contrast, T482 C was more accessible to MTSET in choline buffer at negative holding potent ials, but there was no effect of voltage in sodium buffer. In conclusion, T MD-9 may be involved in transducing conformational changes between the cati on-binding sites and the substrate-binding site in NaDC-1, and it may also form part of the translocation pathway through the transporter.