title: Control of serotonin transporter phosphorylation by conformational state.
abstract: Serotonin transporter (SERT) is responsible for reuptake and recycling of 5-hydroxytryptamine (5-HT; serotonin) after its exocytotic release during neurotransmission. Mutations in human SERT are associated with psychiatric disorders and autism. Some of these mutations affect the regulation of SERT activity by cGMP-dependent phosphorylation. Here we provide direct evidence that this phosphorylation occurs at Thr276, predicted to lie near the cytoplasmic end of transmembrane helix 5 (TM5). Using membranes from HeLa cells expressing SERT and intact rat basophilic leukemia cells, we show that agents such as Na(+) and cocaine that stabilize outward-open conformations of SERT decreased phosphorylation and agents that stabilize inward-open conformations (e.g., 5-HT, ibogaine) increased phosphorylation. The opposing effects of the inhibitors cocaine and ibogaine were each reversed by an excess of the other inhibitor. Inhibition of phosphorylation by Na(+) and stimulation by ibogaine occurred at concentrations that induced outward opening and inward opening, respectively, as measured by the accessibility of cysteine residues in the extracellular and cytoplasmic permeation pathways, respectively. The results are consistent with a mechanism of SERT regulation that is activated by the transport of 5-HT, which increases the level of inward-open SERT and may lead to unwinding of the TM5 helix to allow phosphorylation.
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520.
authors.forename: Yuan-Wei
authors.initials: YW
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520.
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Turk
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520 gary.rudnick@yale.edu.
authors.forename: Gary
authors.identifier: 0000-0002-7622-4110
authors.initials: G
authors.lastname: Rudnick
mesh_terms: D001692:Biological Transport; D003042:Cocaine / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007050:Ibogaine / Q000494:pharmacology; D010766:Phosphorylation; D000072756:Protein Conformation, alpha-Helical; D011499:Protein Processing, Post-Translational; D012701:Serotonin / Q000494:pharmacology; D050486:Serotonin Plasma Membrane Transport Proteins / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D050486:Serotonin Plasma Membrane Transport Proteins; D012701:Serotonin; D007050:Ibogaine; D017869:Cyclic GMP-Dependent Protein Kinases; D003042:Cocaine
keywords: conformation; ibogaine; phosphorylation; serotonin; transporter