Huang, J.; Backer, A.; Uchendu, S.; Bekele, B.; Chen, Q.; Orabi, E.; Stix, R.; Zhang, Y.-W.; Rudnick, G.; Hellsberg, E.; Forrest, L. R.

The human serotonin transporter SERT facilitates serotonin (5-HT+) transport into cells by coupling to Na+ co-transport and K+ exchange. Although extracellular Cl- is also essential for transport, whether Cl- ions are transported has been disputed, raising the question why Cl- ions are required? Here, we examine the role of Cl- using transport measurements, conformational assays, and molecular simulations. We show that Cl- is not transported and does not affect Na+-mediated cytoplasmic pathway closure but does reduce the accessibility of residues in the extracellular pathway, mimicking transport-related occlusion. Simulations indicate that Cl- ion binding constrains the helices in the so-called bundle but not interactions spanning the extracellular pathway thought to act as a molecular gate. We surmise that Cl- (i) increases the stability of surrounding helices, (ii) enhances Na+ binding affinity, and (iii) decreases extracellular pathway accessibility, thereby facilitating transport-related conformational changes. These findings explain SERT's requirement for chloride and highlight distinct features of proteins in the same neurotransmitter transporter family.