Mapping Functionally Important Residues in the Na+/Dicarboxylate Cotransporter, NaDC1

Biochemistry. 2017 Aug 22;56(33):4432-4441. doi: 10.1021/acs.biochem.7b00503. Epub 2017 Aug 10.


Transporters from the SLC13 family couple the transport of two to four Na+ ions with a di- or tricarboxylate, such as succinate or citrate. We have previously modeled mammalian members of the SLC13 family, including the Na+/dicarboxylate cotransporter NaDC1 (SLC13A2), based on a structure of the bacterial homologue VcINDY in an inward-facing conformation with one sodium ion bound at the Na1 site. In the study presented here, we modeled the outward-facing conformation of rabbit and human NaDC1 (rbNaDC1 and hNaDC1, respectively) using an outward-facing model of VcINDY as a template and identified residues in or near the putative Na2 and Na3 cation binding sites. Guided by the structural models in both conformations, we performed site-directed mutagenesis in rbNaDC1 for residues proposed to be in the Na+ or substrate binding sites. Cysteine substitution of T474 in the predicted Na2 binding site results in an inactive protein. The M539C mutant has a low apparent affinity for both sodium and lithium cations, suggesting that M539 may form part of the putative Na3 binding site. The Y432C and T86C mutants have increased Km values for succinate, supporting their proposed location in the outward-facing substrate binding site. In addition, cysteine labeling by MTSEA-biotin shows that Y432C is accessible from the outside of the cell, and the accessibility changes in the presence or absence of Na+. The results of this study improve our understanding of substrate and ion recognition in the mammalian members of the SLC13 family and provide a framework for developing conformationally specific inhibitors against these transporters.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Binding Sites
  • Cations, Monovalent / chemistry
  • Cations, Monovalent / metabolism
  • Dicarboxylic Acid Transporters / chemistry*
  • Dicarboxylic Acid Transporters / genetics
  • Dicarboxylic Acid Transporters / metabolism
  • HEK293 Cells
  • Humans
  • Lithium / chemistry*
  • Lithium / metabolism
  • Models, Molecular*
  • Mutation, Missense
  • Organic Anion Transporters, Sodium-Dependent / chemistry*
  • Organic Anion Transporters, Sodium-Dependent / genetics
  • Organic Anion Transporters, Sodium-Dependent / metabolism
  • Peptide Mapping
  • Rabbits
  • Sodium / chemistry*
  • Sodium / metabolism
  • Succinic Acid / chemistry*
  • Succinic Acid / metabolism
  • Symporters / chemistry*
  • Symporters / genetics
  • Symporters / metabolism


  • Cations, Monovalent
  • Dicarboxylic Acid Transporters
  • Organic Anion Transporters, Sodium-Dependent
  • SLC13A2 protein, human
  • Symporters
  • Lithium
  • Sodium
  • Succinic Acid