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

Abstract

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.

Phosphoinositides are concentrated within the inner leaflets of plasma membranes where they serve as membrane anchors for cytoplasmic proteins and as signaling molecules (1). Phosphatidylinositol-4,5-bisphosphate (PIP2) is produced by 1-phosphatidylinositol 4-kinase (PI-4 kinase) and PIP-5 kinase through the sequential phosphorylation of phosphatidylinositol. PIP2 is the precursor of two important second messengers: inositol triphosphate (IP3) and diacylglycerol [DAG (2)]. These two PIP2 products result from cleavage by phospholipase C (PLC) that is activated via receptors coupled to PLC. IP3stimulates Ca2+ release from the endoplasmic reticulum, whereas DAG directly activates most of the known protein kinase C isoforms. However, PIP2 is not only a precursor for second messengers but is also by itself important for signaling as it serves as an anchor for protein kinase C (3), directly binds to various membrane proteins (1), and is enriched in membrane rafts (4). Thereby, PIP2 controls essential functions of neurons, such as exocytosis (5), endocytosis (6), and transmembrane ion fluxes (7, 8).

Ion fluxes have also been observed in neurotransmitter:sodium symporters (NSSs). Members of this protein family, such as the transporters for dopamine (DAT), norepinephrine (NET), serotonin (5HT; SERT), and GABA (GAT), mediate uptake of released neurotransmitters from the synaptic cleft. In addition, they are targets for addictive drugs such as amphetamines (9). Ion fluxes through these proteins are required for amphetamine-evoked substrate efflux but not for transmitter reuptake (10).

In contrast to neuronal ion channels and ion transporters, NSSs have not been reported to be regulated by PIP2, although other plasma membrane constituents such as cholesterol are required for the proper function of SERT (11⇓–13) and DAT (14). Herein, we reveal SERT as a unique binding partner of membrane PIP2, characterize the binding site involved, and show that PIP2 is necessary for amphetamine actions but not for substrate reuptake. By targeting this interaction, one could thus prevent the contribution of SERT to addiction without affecting its physiological function.