Conformationally sensitive residues in transmembrane domain 9 of the Na+/dicarboxylate co-transporter.
The Na(+)/dicarboxylate co-transporter, NaDC-1, couples the transport of sodium and Krebs cycle intermediates, such as succinate and citrate. Previous studies identified two functionally important amino acids, Glu-475 and Cys-476, 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)ethyl]methanethiosulfonate (MTSET) to the replacement cysteines was determined. Cysteine substitution was tolerated at all but five of the sites: the A461C mutant was not present at the plasma membrane, whereas the F473C, T474C, E475C, and N479C mutants were inactive proteins located on the plasma membrane. Cysteine substitution of four residues found near the extracellular surface of TMD-9 (Ser-478, Ala-480, Ala-481, and Thr-482) resulted in proteins that were sensitive to inhibition by MTSET. The accessibility of MTSET to the four substituted cysteines was highest in the presence of the transported cations, sodium or lithium, and low in choline. The four mutants also exhibited substrate protection of MTSET accessibility. The MTSET accessibility to S478C, A481C, and A480C was independent of voltage. In contrast, T482C was more accessible to MTSET in choline buffer at negative holding potentials, but there was no effect of voltage in sodium buffer. In conclusion, TMD-9 may be involved in transducing conformational changes between the cation-binding sites and the substrate-binding site in NaDC-1, and it may also form part of the translocation pathway through the transporter.
Pajor
A M
AM
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555, USA. ampajor@utmb.edu
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry*; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D029382:Dicarboxylic Acid Transporters; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D007202:Indicators and Reagents / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D008698:Mesylates / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism; D029363:Organic Anion Transporters, Sodium-Dependent; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D027981:Symporters; D013997:Time Factors; D014981:Xenopus
D002352:Carrier Proteins; D002412:Cations; D029382:Dicarboxylic Acid Transporters; D007202:Indicators and Reagents; D008565:Membrane Proteins; D008698:Mesylates; D009841:Oligonucleotides; D029363:Organic Anion Transporters, Sodium-Dependent; C092524:SLC13A2 protein, human; D027981:Symporters; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D003545:Cysteine