Format

Send to

Choose Destination
J Biol Chem. 2015 May 22;290(21):13263-78. doi: 10.1074/jbc.M115.641357. Epub 2015 Apr 13.

A salt bridge linking the first intracellular loop with the C terminus facilitates the folding of the serotonin transporter.

Author information

1
From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and.
2
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.
3
From the Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, A-1090 Vienna, Austria and sonja.sucic@meduniwien.ac.at.

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.

KEYWORDS:

amphipathic helix; dopamine transporter; endoplasmic reticulum (ER); pharmacochaperoning; protein folding; serotonin transporter; trafficking

PMID:
25869136
PMCID:
PMC4505579
DOI:
10.1074/jbc.M115.641357
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center