title: Transmembrane helix 7 in the Na+/dicarboxylate cotransporter 1 is an outer helix that contains residues critical for function.
abstract: Citric acid cycle intermediates, including succinate and citrate, are absorbed across the apical membrane by the NaDC1 Na+/dicarboxylate cotransporter located in the kidney and small intestine. The secondary structure model of NaDC1 contains 11 transmembrane helices (TM). TM7 was shown previously to contain determinants of citrate affinity, and Arg-349 at the extracellular end of the helix is required for transport. The present study involved cysteine scanning mutagenesis of 26 amino acids in TM7 and the associated loops. All of the mutants were well expressed on the plasma membrane, but many had low or no transport activity: 6 were inactive and 7 had activity less than 25% of the parental. Three of the mutants had notable changes in functional properties. F336C had increased transport activity due to an increased Vmax for succinate. The conserved residue F339C had very low transport activity and a change in substrate selectivity. G356C in the putative extracellular loop was the only cysteine mutant that was affected by the membrane-impermeant cysteine reagent, MTSET. However, direct labeling of G356C with MTSEA-biotin gave a weak signal, indicating that this residue is not readily accessible to more bulky reagents. The results suggest that the amino acids of TM7 are functionally important because their replacement by cysteine had large effects on transport activity. However, most of TM7 does not appear to be accessible to the extracellular fluid and is likely to be an outer helix in contact with the lipid bilayer.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California-San Diego, La Jolla, CA 92093-0718, USA. apajor@ucsd.edu
authors.forename: Ana M
authors.initials: AM
authors.lastname: Pajor
authors.forename: Nina N
authors.initials: NN
authors.lastname: Sun
authors.forename: Aditya D
authors.initials: AD
authors.lastname: Joshi
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Randolph
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D029382:Dicarboxylic Acid Transporters / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008698:Mesylates; D008969:Molecular Sequence Data; D009154:Mutation; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D019802:Succinic Acid / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000502:physiology
chemical_list: D029382:Dicarboxylic Acid Transporters; D008698:Mesylates; D029363:Organic Anion Transporters, Sodium-Dependent; D013439:Sulfhydryl Reagents; D027981:Symporters; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D019802:Succinic Acid