A Kinetic Description of Cytosolic K+ Binding to the Human Sertotonin Transporter Under Turnover Conditions

Abstract

The human serotonin transporter (hSERT) belongs to the neurotransmitter:sodium symporter (NSS) family, which regulate neurotransmission by re-uptaking the released neurotransmitters into the presynaptic neuron. During the transport cycle, the transporter alternates between the outward-facing (OF) and inward-facing (IF) states to translocate the substrate across the membrane. Central to the cycle in hSERT is the OF-to-IF state transition, during which 5-HT (serotonin) is inwardly symported in a Na+- and Cl--dependent manner, followed by cytosolic-K+-binding-triggered returning to the OF state. K+ binding might act as a “kinetic decision point” by frustrating the outward transport of 5-HT, namely, the reverse of the physiological direction. However, the mechanism of such K+ regulation remains elusive. Moreover, the conventional transport stoichiometry (15-HTin:1Na+in:1Cl−in:1K+out) has been challenged by recent studies showing that Cl- might remain bound to hSERT during the entire cycle. To explore the role of cytosolic K+ binding to IF hSERT, we have constructed a Markov State Model from an extensive set of molecular dynamics simulations. Starting with the IF state, the system is modeled under turnover conditions, i.e., post-5-HT-release and exposed to cytosolic K+ concentration, resulting in 50 independent, 200-ns replicas. More than half of the trajectories capture spontaneous K+ binding to the Na2 site, a conserved Na+ binding site within NSSs deemed to control the transporter conformational changes and substrate release. Furthermore, consistent with recent experimental results, Cl- was found to remain bound in the majority of the ensembles, questioning the contribution of the Cl- gradient to the substrate transport. Together, these results characterize the previously unknown K+ site in hSERT and present a quantitative kinetic description of K+ binding, shedding light on the mechanism of the ion-dependence of the IF-to-OF transition that resets the transport cycle.