PIP2 mediated influence on Serotonin Transport Function

The serotonin transporter (SERT) plays a key function in the termination of serotonergic neurotransmission. SERT is the main pharmacological target in treating depressive disorders and also a target for various drugs of abuse. Drugs like amphetamine‐type stimulants (ATS) reverse the direction of transport which finally leads to an increased serotonin concentration in the synaptic cleft. Transmembrane proteins get in close contact with the lipid environment and are partitioned in specific lipid microdomains. In a previous study we already implicated the phosphatidylinositol PIP2, a major signaling molecule, to influence amphetamine effects at SERT and elucidated a specific binding interface (Buchmayer, Schicker et al. 2013). We explored a positively charged SERT area using a computational approach and identified a putative second binding site which is close to the inner leaflet of the plasma membrane. Moreover by using single molecule analysis we could show that this interaction stabilizes the oligomeric state of SERT. Disruption of the PIP2 binding site on SERT as well as depletion of PIP2 at the plasma membrane resulted in decreased oligomerization which was also examined using single molecule fluorescence microscopy.ResultsManipulating cellular PIP2 levels had an effect on amphetamine‐induced substrate efflux. Neutralizing of this positively charged SERT area led to a loss of this effect.DiscussionWe could show that both binding sites are necessary for a stable PIP2‐SERT interaction. Neutralization of positive charges within the binding sites abolished PIP2 modulation of amphetamine induced efflux. By drastically reducing intracellular PIP2 levels we could show an decreased amphetamine induced efflux in SERT. This effect could not be observed in mutant SERT indicating a loss of PIP2 mediated effect on substrate efflux. Further we could show that SERT not only interacts with PIP2 but also other phosphatidylinositol species. This interaction is almost lost upon neutralization of both binding sites.Support or Funding InformationThe work of was financially supported by the Austrian Science Foundation/FWF