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

Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP₂) 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 PIP₂ 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.