An Ionic Lock Between Intracellular Loop 1 and a C‐terminal Amphipathic Helix Stabilizes the Serotonin Transporter Along its Folding Trajectory

Abstract

The serotonin transporter (SERT) terminates neurotransmission via rapid reuptake of serotonin from the synaptic cleft. ER export of solute carrier‐6 (SLC6) family members relies on the recruitment of the COPII coat. The COPII component SEC24C binds to an ER export motif located on SERT C‐terminus. We here describe a C‐terminal amphipathic helix that plays a key role in SERT folding. Non‐conservative mutations in the hydrophobic rim of the helix lead to ER retention. Defective ER export of mutant SERTs is not caused by loss of SEC24C binding, revealed by siRNA‐induced SEC24 depletion. ER export of SERT favors inward‐facing transporter conformations. Treatment with the pharmacochaperone noribogaine, which preferentially binds to and stabilizes inward‐facing conformations, recovered the surface expression of C‐terminal helix mutants. Introducing second site suppressor mutations that promote inward‐facing states also repaired their targeting to the membrane. We also probed ionic interactions between the C‐terminus and the first intracellular loop of SERT. Charge‐reversal mutations revealed an ionic lock between E615 in the C‐terminus and R152 in the first intracellular loop. Reversing the charge of each residue alone (R152E or E615K) dramatically reduced SERT surface expression. Conversely, a tandem charge‐reversal mutant (R152E‐E615K) fully restored SERT cell surface expression. We here propose a mechanistic model where the C‐terminal amphipathic helix is stabilized by an intramolecular ionic lock with the first intracellular loop, to support the folding trajectory of SERT and other SLC6 transportersThis work is supported by the Austrian Science Fund Grant P27518‐B27.