Biology subjects

Hellsberg, E.

Publications and source records attributed to Hellsberg, E..

3 recordsLinked to original sources

bioRxiv2025

The Role of Chloride Ions in Serotonin Transport

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 statementThe 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.

biochemistry↗

bioRxiv2023

Identification of the potassium binding site in serotonin transporter SERT

Clearance of serotonin (5-hydroxytryptamine, 5-HT) from the synaptic cleft after neuronal signaling is mediated by serotonin transporter SERT, which couples this process to the movement of a Na+ ion down its chemical gradient. After release of 5-HT and Na+ into the cytoplasm, the transporter faces a rate-limiting challenge of resetting its conformation to be primed again for 5-HT and Na+ binding. Early studies of vesicles containing native SERT revealed that K+ gradients can provide an additional driving force, via K+ antiport. Moreover, under appropriate conditions, a H+ ion can replace K+. Intracellular K+ accelerates the resetting step. Structural studies of SERT have identified two binding sites for Na+ ions, but the K+ site remains enigmatic. Here, we show that K+ antiport can drive substrate accumulation into vesicles containing SERT extracted from a heterologous expression system, allowing us to study the residues responsible for K+ binding. To identify candidate binding residues, we examine many cation binding configurations using molecular dynamics simulations, predicting that K+ binds to the so- called Na2 site. Site directed mutagenesis of residues in this site can eliminate the ability of both K+ and H+ to drive 5-HT accumulation into vesicles and, in patch clamp recordings, prevent the acceleration of turnover rates and the formation of a channel-like state by K+ or H+. In conclusion, the Na2 site plays a pivotal role in orchestrating the sequential binding of Na+ and then K+ (or H+) ions to facilitate 5-HT uptake in SERT. Significance statementNeuronal signaling depends on efficient clearance of the neurotransmitter from the synaptic cleft. To this end, proteins such as serotonin transporter (SERT) leverage the gradients of Na+ and K+ ions across the cell membrane, generated by Na+/K+-ATPase. While the role of Na+ in neurotransmitter transport is well understood, our understanding of the role of potassium in SERT has been limited. In this study, the authors use a combination of biochemical, electrophysiological, and computational tools, to identify the Na2 site as the binding site for K+, shedding light on a critical aspect of neurotransmitter transport.

biochemistry↗

bioRxiv2019

A structural model of the human serotonin transporter in an outward-occluded state

The human serotonin transporter hSERT facilitates the reuptake of its endogenous substrate serotonin from the synaptic cleft into presynaptic neurons after signaling. Reuptake regulates the availability of this neurotransmitter and therefore hSERT plays an important role in balancing human mood conditions. In 2016, the first 3D structures of this membrane transporter were reported in an inhibitor-bound, outward-open conformation. These structures revealed valuable information about interactions of hSERT with antidepressant drugs. Nevertheless, the question remains how serotonin facilitates the specific conformational changes that open and close pathways from the synapse and to the cytoplasm as required for transport. Here, we present a serotonin-bound homology model of hSERT in an outward-occluded state, a key intermediate in the physiological cycle, in which the interactions with the substrate are likely to be optimal. Our approach uses two template structures and includes careful refinement and comprehensive computational validation. According to microsecond-long molecular dynamics simulations, this model exhibits interactions between the gating residues in the extracellular pathway, and these interactions differ from those in an outward-open conformation of hSERT bound to serotonin. Moreover, we predict several features of this state by monitoring the intracellular gating residues, the extent of hydration, and, most importantly, protein-ligand interactions in the central binding site. The results illustrate common and distinct characteristics of these two transporter states and provide a starting point for future investigations of the transport mechanism in hSERT.

biophysics↗