Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog

Sotiria Tavoulari, Eleonora Margheritis, Anu Nagarajan, David C DeWitt, Yuan-Wei Zhang, Edwin Rosado, Silvia Ravera, Elizabeth Rhoades, Lucy R Forrest, Gary Rudnick

Research output: Contribution to journalArticlepeer-review


In LeuT, a prokaryotic homolog of neurotransmitter transporters, Na(+) stabilizes outward-open conformational states. We examined how each of the two LeuT Na(+) binding sites contributes to Na(+)-dependent closure of the cytoplasmic pathway using biochemical and biophysical assays of conformation. Mutating either of two residues that contribute to the Na2 site completely prevented cytoplasmic closure in response to Na(+), suggesting that Na2 is essential for this conformational change, whereas Na1 mutants retained Na(+) responsiveness. However, mutation of Na1 residues also influenced the Na(+)-dependent conformational change in ways that varied depending on the position mutated. Computational analyses suggest those mutants influence the ability of Na1 binding to hydrate the substrate pathway and perturb an interaction network leading to the extracellular gate. Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na(+) and transmembrane helices 1 and 8, whereas Na(+) binding at Na1 influences conformational change through a network of intermediary interactions. The results also provide evidence that N-terminal release and helix motions represent distinct steps in cytoplasmic pathway opening.

Original languageEnglish
Pages (from-to)1456-71
Number of pages16
JournalThe Journal of biological chemistry
Issue number3
StatePublished - Jan 15 2016


  • Amino Acid Substitution
  • Amino Acid Transport Systems/chemistry
  • Aquatic Organisms/metabolism
  • Bacterial Proteins/chemistry
  • Binding Sites
  • Cysteine/chemistry
  • Gram-Negative Bacteria/metabolism
  • Ligands
  • Liposomes
  • Models, Molecular
  • Molecular Dynamics Simulation
  • Mutagenesis, Site-Directed
  • Mutation
  • Plasma Membrane Neurotransmitter Transport Proteins/chemistry
  • Protein Conformation
  • Protein Folding
  • Protein Stability
  • Proteolipids
  • Recombinant Proteins/chemistry
  • Sodium/metabolism


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