Biology subjects

Boytsov, D.

Publications and source records attributed to Boytsov, D..

3 recordsLinked to original sources

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↗

bioRxiv2022

Structure-based Discovery of Conformationally Selective Inhibitors of the Serotonin Transporter

The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has an unusual anti-depressant profile and stabilizes the inward-open conformation. Unfortunately, ibogaine is promiscuous and cardiotoxic, limiting understanding of inward-open state ligands. We computationally docked over 200 million small molecules against the ibogaine stabilized inward-open state of SERT. Thirty-six top-ranking compounds were synthesized and thirteen inhibited with potencies ranging from 29 to 5000 nM. Structure-based optimization led to two novel inhibitors with Ki values down to 3 nM. The new molecules stabilized an outward-closed state of the transporter and had little activity against off-targets. A cryo-EM structure of one of these bound to SERT confirmed the predicted geometry. In mouse behavioral assays, both had anxiolytic and anti-depressant activity, with potencies up to 200 better than fluoxetine.

biochemistry↗

bioRxiv2022

Trapped pore waters in the open proton channel HV1

The voltage-gated proton channel, HV1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, we estimate the water mobility for the putative case of an uninterrupted wire. The predicted single-channel water permeability 3x10-12cm3s-1 reflects the permeability-governing number of hydrogen bonds between water molecules in single-file configuration and pore residues. However, the measured unitary water permeability does not confirm the prediction, i.e., it is negligible. Osmotic deflation of reconstituted lipid vesicles reveals trapped water inside the HV1 wild-type channel and D174A mutant open at 0 mV. The conductance of 1400 H+ s-1 per wild-type channel agrees with the calculated diffusion limit for a ~2 [A] capture radius for protons. Removal of a charged amino acid (D174) at the pore mouth decreases H+ conductance, conceivably by reducing the capture radius. At least one intervening amino acid contributes to H+ conductance while blocking water flow.

biophysics↗