The Role of Chloride Ions in Serotonin Transport

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.