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

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 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

High-affinity GABA uptake by neuronal GAT1 transporters provokes release of [3H]GABA by homoexchange and through GAT1-independent Ca2+-mediated mechanisms

“…These results strengthen the view that inward and outward transport of biogenic amines are distinct mechanistic phenomena (Robertson et al, 2009;Sitte and Freissmuth, 2010). In contrast, PIP 2 was found unable to regulate substrate efflux through the GABA transporter GAT1, indicating that the results obtained with the amine transporters do not apply to GAT1, although the latter belongs to the neurotransmitter: sodium symporter family (Buchmayer et al, 2013).…”

“…different from the efflux component. A very recent report on the amphetamine actions at SERT demonstrates that phosphatidylinositol-4,5-bisphosphate (PIP 2 ) binding to SERT is required for the amphetamine-evoked SERT-mediated serotonin release, but is not a requirement for serotonin uptake (Buchmayer et al, 2013). These results strengthen the view that inward and outward transport of biogenic amines are distinct mechanistic phenomena (Robertson et al, 2009;Sitte and Freissmuth, 2010).…”

Amphetamine Action at the Cocaine- and Antidepressant-Sensitive Serotonin Transporter Is Modulated by αCaMKII

“…More importantly, these observations and related findings with DAT (Steinkellner et al, 2012 stress the apparent asymmetry of substrate uptake by and efflux through monoamine transporters (Sitte and Freissmuth, 2015). A comparable asymmetry in uptake and efflux has also been found for the interaction of SERT and DAT with phosphoinositides: depletion of phosphatidylinositol-4,5-bisphosphate impaired amphetamine-induced efflux through SERT and DAT, but had no effect on substrate uptake (Buchmayer et al, 2013;Hamilton et al, 2014). These studies imply that substrate uptake and efflux are, at least in part, subject to different regulatory input.…”

“…The cytosolic accumulation of both Na ϩ and substrate allows for reverse transport. However, this simplified model does not take into account the asymmetrical nature of inward and outward transport: amphetamine-triggered reverse transportbut not substrate influx-for example, is contingent on the presence of phosphatidylinositol-4,5-bisphosphate (Buchmayer et al, 2013). Interestingly, calmodulin kinase II␣ (␣CaMKII) was found to play a critical role in amphetamine-triggered reverse transport of dopamine by DAT via an interaction of the kinase with the C terminus of the transporter (Fog et al, 2006;Rickhag et al, 2013).…”

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

“…One way in which PI3K may regulate SERT activity is through control of PIP2 levels, which, as with DAT, interacts with the SERT N terminus and regulates PCA-induced 5-HT efflux (Buchmayer et al, 2013). Manipulation of this interaction did not appear to affect 5-HT uptake, however.…”