Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate

Florian Buchmayer, Klaus Schicker, Thomas Steinkellner, Petra Geier, Gerald Stübiger, Peter J. Hamilton, Andreas Jurik, Thomas Stockner, Jae Won Yang, Therese Montgomery, Marion Holy, Tina Hofmaier, Oliver Kudlacek, Heinrich J.G. Matthies, Gerhard F. Ecker, Valery Bochkov, Aurelio Galli, Stefan Boehm, Harald H. Sitte

Research output: Contribution to journalArticlepeer-review

53 Scopus citations

Abstract

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 PIP2 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 PIP2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP2-synthesis. We tested the effects of the verified scarcity of PIP2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP2 in pull-down assays. On decreased PIP2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamine-induced 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 PIP2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP2. Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP2-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.

Original languageEnglish
Pages (from-to)11642-11647
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume110
Issue number28
DOIs
StatePublished - 9 Jul 2013
Externally publishedYes

Keywords

  • Amperometry
  • Mass spectrometry
  • Phosphoinositide
  • Release
  • Reuptake

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