Intramolecular cross-linking in a bacterial homolog of mammalian SLC6 neurotransmitter transporters suggests an evolutionary conserved role of transmembrane segments 7 and 8

Julie Kniazeff, Claus Juul Loland, Naomi Goldberg, Matthias Quick, Shonit Das, Harald H Sitte, Jonathan A Javitch, Ulrik Gether

Research output: Contribution to journalJournal articleResearchpeer-review

9 Citations (Scopus)

Abstract

The extracellular concentration of the neurotransmitters dopamine, serotonin, norepinephrine, GABA and glycine is tightly controlled by plasma membrane transporters belonging to the SLC6 gene family. A very large number of putative transport proteins with a remarkable homology to the SLC6 transporters has recently been identified in prokaryotes. Here we have probed structural relationships in a 'microdoman' corresponding to the extracellular ends of transmembrane segments (TM) 7 and 8 in one of these homologs, the tryptophan transporter TnaT from Symbiobacterium thermophilum. We found that simultaneous - but not individual - substitution of Ala286 at the top of TM7 and Met311 at the top of TM8 with cysteines conferred sensitivity to submicromolar concentrations of Hg(2+) as assessed in a [(3)H]tryptophan uptake assay. Because Hg(2+) can cross-link pairs of cysteines, this suggests close proximity between TM 7 and 8 in the tertiary structure of TnaT as previously suggested for the mammalian counterparts. Furthermore, the inhibition of uptake upon cross-linking the two cysteines provides indirect support for a conserved conformational role of these transmembrane domains in the transport process. It was not possible, however, to transfer to TnaT binding sites for another metal ion, Zn(2+), that we previously engineered in the dopamine (DAT) and GABA (GAT-1) transporters between TM 7 and 8. This suggests that the structure of the TM7/8 microdomain is not identical with that of DAT and GAT-1. Hence, our data also emphasize possible structural differences that should be taken into account when interpreting future data on bacterial homologs of the SLC6 transporters.
Original languageEnglish
JournalNeuropharmacology
Volume49
Issue number6
Pages (from-to)715-23
Number of pages9
ISSN0028-3908
DOIs
Publication statusPublished - Nov 2005

Keywords

  • Animals
  • Biological Evolution
  • Cross-Linking Reagents
  • Dose-Response Relationship, Drug
  • Escherichia coli
  • Extracellular Space
  • Gene Expression
  • Mammals
  • Membrane Transport Proteins
  • Mercury
  • Mesylates
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Protein Structure, Quaternary
  • Sequence Homology, Amino Acid
  • Structure-Activity Relationship
  • Tritium
  • Tryptophan

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