Cytosolic K⁺ Binding to the Human Serotonin Transporter

The human serotonin transporter (hSERT) is a member of the neurotransmitter:sodium symporter (NSS) family that mediates active reuptake of serotonin from the synapse into the presynaptic neuron. During the transport cycle, hSERT alternates between outward-facing (OF) and inward-facing (IF) states to translocate its substrate between the two sides of the membrane. During the OF-to-IF state transition, serotonin (aka, 5-HT) is inwardly symported together with Na⁺ and Cl^- ions. The return to the OF state is facilitated by cytosolic K⁺ binding, a step that is also proposed to act as a kinetic decision point by frustrating the outward transport of 5-HT in the direction opposite to the physiological direction of the cycle. However, as opposed to the Na⁺ ions, the mechanism of K⁺ binding, its binding site and regulation have not been thoroughly studied. Moreover, recent studies have challenged the conventional transport stoichiometry (1 5-HT_in:1 Na_in⁺:1 Cl_in^-:1 K_out⁺) in hSERT, suggesting that Cl^- might remain bound to the transporter during the entire cycle. To explore the role of cytosolic K⁺ binding to IF hSERT, we performed an extensive set of molecular dynamics simulations. Starting from the post-release IF conformation and in the presence of cytosolic K⁺, we generated 50 independent trajectories, each for 200 ns to study the behavior of ions. In more than half of the simulations, spontaneous K⁺ binding was observed at the Na2 site, a conserved cation-binding site in NSS transporters that has been implicated in controlling conformational transitions. Markov state model analysis of coupled ion dynamics quantifies K⁺ binding kinetics and identifies K⁺ occupancy of the Na2 site, with Na⁺ retained at Na1, as the thermodynamically dominant post-release state. In addition, Cl^- remains bound to hSERT in the majority of sampled simulations, consistent with recent experimental observations and suggesting a limited role of Cl^- release during this stage of the transport cycle. Together, these results provide a kinetic and mechanistic framework for understanding cytosolic K⁺ binding to hSERT and its potential role in facilitating the IF-to-OF transition that resets the transport cycle.