title: A salt bridge linking the first intracellular loop with the C terminus facilitates the folding of the serotonin transporter.
abstract: The folding trajectory of solute carrier 6 (SLC6) family members is of interest because point mutations result in misfolding and thus cause clinically relevant phenotypes in people. Here we examined the contribution of the C terminus in supporting folding of the serotonin transporter (SERT; SLC6A4). Our working hypothesis posited that the amphipathic nature of the C-terminal α-helix (Thr(603)-Thr(613)) was important for folding of SERT. Accordingly, we disrupted the hydrophobic moment of the α-helix by replacing Phe(604), Ile(608), or Ile(612) by Gln. The bulk of the resulting mutants SERT-F604Q, SERT-I608Q, and SERT-I612Q were retained in the endoplasmic reticulum, but their residual delivery to the cell surface still depended on SEC24C. This indicates that the amphipathic nature of the C-terminal α-helix was dispensable to endoplasmic reticulum export. The folding trajectory of SERT is thought to proceed through the inward facing conformation. Consistent with this conjecture, cell surface expression of the misfolded mutants was restored by (i) introducing second site suppressor mutations, which trap SERT in the inward facing state, or (ii) by the pharmacochaperone noribogaine, which binds to the inward facing conformation. Finally, mutation of Glu(615) at the end of the C-terminal α-helix to Lys reduced surface expression of SERT-E615K. A charge reversal mutation in intracellular loop 1 restored surface expression of SERT-R152E/E615K to wild type levels. These observations support a mechanistic model where the C-terminal amphipathic helix is stabilized by an intramolecular salt bridge between residues Glu(615) and Arg(152). This interaction acts as a pivot in the conformational search associated with folding of SERT.
authors: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and.
authors: Florian
authors: F
authors: Koban
authors: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and the Department of Pharmacology, Faculty of Veterinary Medicine, Mansoura University, 35516 Mansoura, Egypt.
authors: Ali
authors: A
authors: El-Kasaby
authors: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and.
authors: Cornelia
authors: C
authors: Häusler
authors: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and.
authors: Thomas
authors: T
authors: Stockner
authors: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and.
authors: Benedikt M
authors: BM
authors: Simbrunner
authors: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and.
authors: Harald H
authors: HH
authors: Sitte
authors: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and.
authors: Michael
authors: M
authors: Freissmuth
authors: From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and sonja.sucic@meduniwien.ac.at.
authors: Sonja
authors: S
authors: Sucic
mesh_terms: D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D018613:Microscopy, Confocal; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D050486:Serotonin Plasma Membrane Transport Proteins
keywords: amphipathic helix; dopamine transporter; endoplasmic reticulum (ER); pharmacochaperoning; protein folding; serotonin transporter; trafficking