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

Buchmayer, Florian, Schicker, Klaus, Steinkellner, Thomas, Geier, Petra, Stübiger, Gerald, Hamilton, Peter J., Jurik, Andreas, Stockner, Thomas, Yang, Jae-Won, Montgomery, Therese, Holy, Marion, Hofmaier, Tina, Kudlacek, Oliver, Matthies, Heinrich J. G., Ecker, Gerhard F., Bochkov, Valery, Galli, Aurelio, Boehm, Stefan, Sitte, Harald H.
Proceedings of the National Academy of Sciences of the United States of America 2013 v.110 no.28 pp. 11642-11647
amphetamine, binding sites, extracellular space, humans, ion channels, mutants, mutation, nerve tissue, norepinephrine, phospholipase C, rats, serotonin, transporters
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 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 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.