A salt bridge linking the first intracellular loop with the C-terminus facilitates the folding of the serotonin transporter [Neurobiology]

April 13th, 2015 by Koban, F., El–Kasaby, A., Hausler, C., Stockner, T., Simbrunner, B. M., Sitte, H. H., Freissmuth, M., Sucic, S.

The folding trajectory of SLC6 (solute carrier 6) 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 (T603 to T613) was important for folding of SERT. Accordingly, we disrupted the hydrophobic moment of the α-helix by replacing F604, I608 or I612 by Q. The bulk of the resulting mutants SERT-F604Q, SERT-I608Q and SERT-I612Q was retained in the endoplasmic reticulum (ER), 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 ER 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 E615 at the end of the C-terminal α-helix to K 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 E615 and R152. This interaction acts as a pivot in the conformational search associated with folding of SERT.