Ligand effects on cross-linking support a conformational mechanism for serotonin transport.
Serotonin transporter (SERT) is responsible for the re-uptake of 5-hydroxytryptamine (5-HT) from the synaptic cleft after release from serotonergic neurons. We show here that cysteine residues at positions in transmembranes 1 and 3 of SERT, like the corresponding positions in the gamma-aminobutyric acid transporter, can be cross-linked using copper(II)(1,10-phenanthroline)(3). The presence of a cross-link was detected by a novel methionine mutagenesis strategy. A change in mobility for an N-terminal cyanogen bromide fragment accompanied disulfide cross-linking of the two cysteine residues. Cross-linking also inhibited transport, and this process was blocked by cocaine, which is expected to stabilize SERT in conformations where the two positions are separated, but cocaine did not decrease accessibility of either of the two cysteines to modification by 2-aminoethyl methanethiosulfonate. Cysteine was required at both positions on the same molecule for efficient cross-linking, indicating that the reaction was intramolecular.
Tao
Zhen
Z
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, USA.
Zhang
Yuan-Wei
YW
Agyiri
Alice
A
Rudnick
Gary
G
D001692:Biological Transport; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology*; D003488:Cyanogen Bromide / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry; D009154:Mutation; D011487:Protein Conformation; D012701:Serotonin / Q000737:chemistry*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry*
D003432:Cross-Linking Reagents; D008024:Ligands; D026901:Membrane Transport Proteins; D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin; D003545:Cysteine; D003488:Cyanogen Bromide