Biology subjects

Sandtner, W.

Publications and source records attributed to Sandtner, W..

5 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

A mechanism of uncompetitive inhibition of the serotonin transporter

The serotonin transporter (SERT/SLC6A4) is arguably the most extensively studied solute carrier (SLC). During its eponymous action - i.e., the retrieval of serotonin from the extracellular space - SERT undergoes a conformational cycle. Typical inhibitors (antidepressant drugs and cocaine), partial and full substrates (amphetamines and their derivatives) and atypical inhibitors (ibogaine analogues) bind preferentially to different states in this cycle. This results in competitive or non-competitive transport inhibition. Here, we explored the action of N-formyl-1,3-bis (3,4-methylenedioxyphenyl)-prop-2-yl-amine (ECSI#6) on SERT: inhibition of serotonin uptake by ECSI#6 was enhanced with increasing serotonin concentration. Conversely, the KM for serotonin was lowered by augmenting ECSI#6. ECSI#6 bound with low affinity to the outward-facing state of SERT but with increased affinity to a potassium-bound state. Electrophysiological recordings showed that ECSI#6 preferentially interacted with the inward-facing state. Kinetic modeling recapitulated the experimental data and verified that uncompetitive inhibition arose from preferential binding of ECSI#6 to the K+-bound, inward-facing conformation of SERT. This binding mode predicted a pharmacochaperoning action of ECSI#6, which was confirmed by examining its effect on the folding-deficient mutant SERT-PG601,602AA: pre-incubation of HEK293 cells with ECSI#6 restored export of SERT-PG601,602AA from the endoplasmic reticulum and substrate transport. Similarly, in transgenic flies, administration of ECSI#6 promoted delivery of SERT-PG601,602AA to the presynaptic specialization of serotonergic neurons. To the best of our knowledge, ECSI#6 is the first example of an uncompetitive SLC inhibitor. Pharmacochaperones endowed with the binding mode of ECSI#6 are attractive, because they can rescue misfolded transporters at concentrations, which cause modest transport inhibition.

pharmacology and toxicology↗

bioRxiv2021

Handling of intracellular K + determines voltage dependence of plasmalemmal monoamine transporter function

The concentrative power of the transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT) is thought to be fueled by the transmembrane Na+ gradient, but it is conceivable that they can also tap other energy sources, e.g. membrane voltage and/or the transmembrane K+ gradient. We address this by recording uptake of endogenous substrates or the fluorescent substrate APP+ ((4-(4-dimethylamino)phenyl-1-methylpyridinium) under voltage control in cells expressing DAT, NET or SERT. We show that DAT and NET differ from SERT in intracellular handling of K+. In DAT and NET, substrate uptake was voltage-dependent due to the transient nature of intracellular K+ binding, which precluded K+ antiport. SERT, however, antiports K+ and achieves voltage-independent transport. Thus, there is a trade-off between maintaining constant uptake and harvesting membrane potential for concentrative power, which we conclude to occur due to subtle differences in the kinetics of co-substrate ion binding in closely related transporters.

biophysics↗

bioRxiv2019

An electrophysiological approach to measure changes in the membrane surface potential in real time

Biological membranes carry fixed charges at their surfaces. These arise primarily from phospholipid head groups. In addition, membrane proteins contribute to the surface potential with their charged residues. Membrane lipids are asymmetrically distributed. Because of this asymmetry the net negative charge at the inner leaflet exceeds that at the outer leaflet. Changes in surface potential are predicted to shape the capacitive properties of the membrane (i.e. the ability of the membrane to store electrical charges). Here, we show that it is possible to detect changes in surface potential by an electrophysiological approach: the analysis of cellular currents relies on assuming that the electrical properties of a cell are faithfully described by a three-element circuit - i.e. the minimal equivalent circuit - comprised of two resistors and one capacitor. However, to account for changes in surface potential it is necessary to add a battery to this circuit connected in series with the capacitor. This extended circuit model predicts that the current response to a square-wave voltage pulse harbors information, which allows for separating the changes in surface potential from a true capacitance change. We interrogated our model by investigating changes in capacitance induced by ligand binding to the serotonin transporter (SERT) and to the glycine transporters (GlyT1 and GlyT2). The experimental observations were consistent with the predictions of the extended circuit. We conclude that ligand-induced changes in surface potential (reflecting the binding event) and in true membrane capacitance (reflecting the concomitant conformational change) can be detected in real time even in instances where they occur simultaneously.\n\nStatement of SignificanceThe plasma membrane of a cell possesses fixed charges on both surfaces. Surface charges play an important role in many biological processes. However, the mechanisms, which regulate the surface charge densities at the plasma membrane, are poorly understood. This is in part due to lack of experimental approaches that allow for detecting changes in surface charges in real time. Here, we show that it is possible to track alterations in the electric potential at the membrane surface with high temporal resolution by an electrophysiological approach. Importantly, the described method allows for discriminating between a change in surface potential and a change in true membrane capacitance (e.g. a change in membrane area), even if these occur in parallel.

biophysics↗

bioRxiv2018

A label-free approach to detect ligand binding to cell surface proteins in real time

Electrophysiological recordings allow for monitoring the operation of proteins with high temporal resolution down to the single molecule level. This technique has been exploited to track either ion flow arising from channel opening or the synchronized movement of charged residues and/or ions within the membrane electric field. Here, we describe a novel type of current by using the serotonin transporter (SERT) as a model. We examined transient currents elicited on rapid application of specific SERT inhibitors. Our analysis shows that these currents originate from ligand binding and not from a conformational change. The Gouy-Chapman model predicts that a ligand-induced elimination/neutralization of surface charge must produce a displacement current and related apparent changes in membrane capacitance. Here we verified these predictions with SERT. Our observations demonstrate that ligand binding to a protein can be monitored in real time and in a label-free manner by recording the membrane capacitance.

biophysics↗