title: Intramolecular cross-linking in a bacterial homolog of mammalian SLC6 neurotransmitter transporters suggests an evolutionary conserved role of transmembrane segments 7 and 8.
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.
authors.affiliation: Molecular Neuropharmacology Group, Department of Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
authors.forename: Julie
authors.initials: J
authors.lastname: Kniazeff
authors.forename: Claus Juul
authors.initials: CJ
authors.lastname: Loland
authors.forename: Naomi
authors.initials: N
authors.lastname: Goldberg
authors.forename: Matthias
authors.initials: M
authors.lastname: Quick
authors.forename: Shonit
authors.initials: S
authors.lastname: Das
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
mesh_terms: D000818:Animals; D005075:Biological Evolution; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli; D005110:Extracellular Space; D015870:Gene Expression; D008322:Mammals; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008628:Mercury / Q000494:pharmacology; D008698:Mesylates / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014316:Tritium / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D026901:Membrane Transport Proteins; D008698:Mesylates; D014316:Tritium; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D014364:Tryptophan; D008628:Mercury