Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP 2 ) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP 2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP 2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP 2 -synthesis. We tested the effects of the verified scarcity of PIP 2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP 2 in pull-down assays. On decreased PIP 2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamineinduced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP 2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP 2 . Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP 2 -dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP 2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.phosphoinositide | reuptake | release | mass spectrometry | amperometry
The human serotonin transporter (hSERT) mediates uptake of serotonin from the synaptic cleft and thereby terminates serotonergic signalling. We have previously found by singlemolecule microscopy that SERT forms stable higher-order oligomers of differing stoichiometry at the plasma membrane of living cells. Here, we report that SERT oligomer assembly at the endoplasmic reticulum (ER) membrane follows a dynamic equilibration process, characterized by rapid exchange of subunits between different oligomers, and by a concentration dependence of the degree of oligomerization. After trafficking to the plasma membrane, however, the SERT stoichiometry is fixed. Stabilization of the oligomeric SERT complexes is mediated by the direct binding to phosphoinositide phosphatidylinositol-4,5-biphosphate (PIP 2 ). The observed spatial decoupling of oligomer formation from the site of oligomer operation provides cells with the ability to define protein quaternary structures independent of protein density at the cell surface.
HighlightsWe quantified adulterants in street drugs sold as cocaine.We analyzed effects of the most common adulterant levamisole, on neurotransmitter transporters.Differences in the selectivity of levamisole can be explained by homology modelling and docking.Aminorex, a metabolite of levamisole, modulates neurotransmitter transporters directly.Depending on the transporter, aminorex acts as a blocker or as a releaser.
Uptake of neurotransmitters by sodium-coupled monoamine transporters of the NSS family is required for termination of synaptic transmission. Transport is tightly regulated by protein–protein interactions involving the small cytoplasmic segments at the amino- and carboxy-terminal ends of the transporter. Although structures of homologues provide information about the transmembrane regions of these transporters, the structural arrangement of the terminal domains remains largely unknown. Here, we combined molecular modeling, biochemical, and biophysical approaches in an iterative manner to investigate the structure of the 82-residue N-terminal and 30-residue C-terminal domains of human serotonin transporter (SERT). Several secondary structures were predicted in these domains, and structural models were built using the Rosetta fragment-based methodology. One-dimensional 1H nuclear magnetic resonance and circular dichroism spectroscopy supported the presence of helical elements in the isolated SERT N-terminal domain. Moreover, introducing helix-breaking residues within those elements altered the fluorescence resonance energy transfer signal between terminal cyan fluorescent protein and yellow fluorescent protein tags attached to full-length SERT, consistent with the notion that the fold of the terminal domains is relatively well-defined. Full-length models of SERT that are consistent with these and published experimental data were generated. The resultant models predict confined loci for the terminal domains and predict that they move apart during the transport-related conformational cycle, as predicted by structures of homologues and by the “rocking bundle” hypothesis, which is consistent with spectroscopic measurements. The models also suggest the nature of binding to regulatory interaction partners. This study provides a structural context for functional and regulatory mechanisms involving SERT terminal domains.
Cocaine is a naturally occurring and illicitly used psychostimulant drug. Cocaine acts at monoaminergic neurotransmitter transporters to block uptake of the monoamines, dopamine, serotonin and norepinephrine. The resulting increase of monoamines in the extracellular space underlies the positively reinforcing effects that cocaine users seek. In turn, this increase in monoamines underlies the development of addiction, and can also result in a number of severe side effects. Currently, cocaine is one of the most common illicit drugs available on the European market. However, cocaine is increasingly sold in impure forms. This trend is driven by cocaine dealers seeking to increase their profit margin by mixing ("cutting") cocaine with numerous other compounds ("adulterants"). Importantly, these undeclared compounds put cocaine consumers at risk, because consumers are not aware of the additional potential threats to their health. This review describes adulterants that have been identified in cocaine sold on the street market. Their typical pharmacological profile and possible reasons why these compounds can be used as cutting agents will be discussed. Since a subset of these adulterants has been found to exert effects similar to cocaine itself, we will discuss levamisole, the most frequently used cocaine cutting agent today, and its metabolite aminorex.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.