Biotinylation of single cysteine mutants of the glutamate transporter GLT-1 from rat brain reveals its unusual topology

Neuron. 1998 Sep;21(3):623-32. doi: 10.1016/s0896-6273(00)80572-3.


In the central nervous system, (Na+ + K+)-coupled glutamate transporters restrict the neurotoxicity of this transmitter and limit the duration of synaptic excitation at some synapses. The various isotransporters exhibit a particularly high homology in an extended hydrophobic domain of ill-defined topology that contains several determinants involved in ion and transmitter binding. Here, we describe the determination of the membrane topology of the cloned astroglial glutamate transporter GLT-1. A series of functional transporters containing single cysteines was engineered. Their topological disposition was determined by using a biotinylated sulfhydryl reagent. The glutamate transporter has eight transmembrane domains long enough to span the membrane as et heiices. Strikingly, between the seventh and eighth domains, a structure reminiscent of a pore loop and an outward-facing hydrophobic linker are positioned.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • ATP-Binding Cassette Transporters / chemistry*
  • ATP-Binding Cassette Transporters / metabolism*
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Amino Acid Transport System X-AG
  • Animals
  • Astrocytes / metabolism*
  • Biological Transport
  • Biotinylation
  • Brain / metabolism*
  • Cloning, Molecular
  • Cysteine*
  • Glycosylation
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Fragments / chemistry
  • Point Mutation
  • Protein Conformation
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism


  • ATP-Binding Cassette Transporters
  • Amino Acid Transport System X-AG
  • Peptide Fragments
  • Recombinant Proteins
  • Cysteine