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

TY - JOUR

T1 - Intramolecular cross-linking in a bacterial homolog of mammalian SLC6 neurotransmitter transporters suggests an evolutionary conserved role of transmembrane segments 7 and 8

AU - Kniazeff, Julie

AU - Loland, Claus Juul

AU - Goldberg, Naomi

AU - Quick, Matthias

AU - Das, Shonit

AU - Sitte, Harald H

AU - Javitch, Jonathan A

AU - Gether, Ulrik

PY - 2005/11

Y1 - 2005/11

N2 - 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.

AB - 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.

KW - Animals

KW - Biological Evolution

KW - Cross-Linking Reagents

KW - Dose-Response Relationship, Drug

KW - Escherichia coli

KW - Extracellular Space

KW - Gene Expression

KW - Mammals

KW - Membrane Transport Proteins

KW - Mercury

KW - Mesylates

KW - Models, Molecular

KW - Mutagenesis, Site-Directed

KW - Protein Binding

KW - Protein Structure, Quaternary

KW - Sequence Homology, Amino Acid

KW - Structure-Activity Relationship

KW - Tritium

KW - Tryptophan

U2 - 10.1016/j.neuropharm.2005.07.003

DO - 10.1016/j.neuropharm.2005.07.003

M3 - Journal article

C2 - 16129457

SN - 0028-3908

VL - 49

SP - 715

EP - 723

JO - Neuropharmacology

JF - Neuropharmacology

IS - 6

ER -