Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters

J Biol Chem. 2015 Nov 27;290(48):28988-96. doi: 10.1074/jbc.M115.682666. Epub 2015 Oct 16.


Crystal structures of the archaeal homologue GltPh have provided important insights into the molecular mechanism of transport of the excitatory neurotransmitter glutamate. Whereas mammalian glutamate transporters can translocate both glutamate and aspartate, GltPh is only one capable of aspartate transport. Most of the amino acid residues that surround the aspartate substrate in the binding pocket of GltPh are highly conserved. However, in the brain transporters, Thr-352 and Met-362 of the reentrant hairpin loop 2 are replaced by the smaller Ala and Thr, respectively. Therefore, we have studied the effects of T352A and M362T on binding and transport of aspartate and glutamate by GltPh. Substrate-dependent intrinsic fluorescence changes were monitored in transporter constructs containing the L130W mutation. GltPh-L130W/T352A exhibited an ~15-fold higher apparent affinity for l-glutamate than the wild type transporter, and the M362T mutation resulted in an increased affinity of ~40-fold. An even larger increase of the apparent affinity for l-glutamate, around 130-fold higher than that of wild type, was observed with the T352A/M362T double mutant. Radioactive uptake experiments show that GltPh-T352A not only transports aspartate but also l-glutamate. Remarkably, GltPh-M362T exhibited l-aspartate but not l-glutamate transport. The double mutant retained the ability to transport l-glutamate, but its kinetic parameters were very similar to those of GltPh-T352A alone. The differential impact of mutation on binding and transport of glutamate suggests that hairpin loop 2 not only plays a role in the selection of the substrate but also in its translocation.

Keywords: glutamate; membrane transporter reconstitution; neurotransmitter transport; site-directed mutagenesis; sodium transport.

Publication types

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

MeSH terms

  • Amino Acid Substitution
  • Aspartic Acid / chemistry*
  • Aspartic Acid / genetics
  • Aspartic Acid / metabolism
  • Brain / metabolism
  • Glutamate Plasma Membrane Transport Proteins / chemistry*
  • Glutamate Plasma Membrane Transport Proteins / genetics
  • Glutamate Plasma Membrane Transport Proteins / metabolism
  • Glutamic Acid / chemistry*
  • Glutamic Acid / genetics
  • Glutamic Acid / metabolism
  • Humans
  • Ion Transport / genetics
  • Mutation, Missense*
  • Nerve Tissue Proteins / chemistry*
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism
  • Protein Structure, Secondary
  • Substrate Specificity / genetics


  • Glutamate Plasma Membrane Transport Proteins
  • Nerve Tissue Proteins
  • Aspartic Acid
  • Glutamic Acid

Associated data

  • PDB/2NWX