The role of chloride ions in serotonin transport

Abstract The human serotonin (5-HT + ) transporter SERT facilitates 5-HT + transport into cells by coupling it to Na + symport and K + antiport. Although extracellular Cl − is also essential for transport, Cl − co-transport has been disputed, raising questions about the role of Cl − ions and why they are required. We show that Cl − gradients do not impact 5-HT + accumulation, indicating that Cl⁻ does not provide a driving force for uptake and arguing against stoichiometric Cl⁻ symport. The presence of Cl − had only a small effect on Na + -mediated cytoplasmic pathway closure but markedly reduced the accessibility of residues in the extracellular pathway, consistent with modulation of the outward-facing states. Simulations illustrate that Cl − interacts strongly with a bound Na + ion and stabilizes helix packing on the extracellular side. We propose that Cl⁻ acts as an essential architectural cofactor by enhancing Na + affinity and interactions between helices, thereby facilitating transport-related conformational transitions. Significance statement The serotonin transporter (SERT) clears serotonin from synapses and is the target of widely used antidepressants. Although chloride ions are known to be required for SERT function, whether chloride is transported together with serotonin has remained unresolved for decades. We show that chloride gradients do not drive serotonin uptake, arguing against chloride symport. Instead, chloride stabilizes specific conformations of the transporter by strengthening sodium binding and extracellular helix packing. These findings redefine chloride not as a transported substrate, but as an architectural cofactor that facilitates conformational transitions required for transport. This reinterpretation reshapes our understanding of ion dependence in SERT and suggests a broader principle: ion requirements in related transporters may reflect structural stabilization rather than obligatory symport.