Transmembrane helix 7 in the Na+/dicarboxylate cotransporter 1 is an outer helix that contains residues critical for function

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)1454-1461
Number of pages8
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1808
Issue number6
DOIs
StatePublished - Jun 1 2011
Externally publishedYes

    Fingerprint

Keywords

  • Cysteine substitution
  • Methanethiosulfonate reagents
  • Site-directed mutagenesis
  • SLC13 family
  • Sodium
  • Succinate

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

Cite this