Phosphatidylinositol 4,5-bisphosphate (PIP(2)) facilitates norepinephrine transporter dimerization and modulates substrate efflux

The plasmalemmal norepinephrine transporter (NET) regulates cardiovascular sympathetic activity by clearing extracellular norepinephrine in the synaptic cleft. Here, we investigate the subunit stoichiometry and function of NET using single-molecule fluorescence microscopy and flux assays. In particular, we show the effect of phosphatidylinositol 4,5-bisphosphate (PIP(2)) on NET oligomerization and efflux. NET forms monomers (~60%) and dimers (~40%) at the plasma membrane. PIP(2) depletion results in a decrease in the average oligomeric state and decreases NET-mediated substrate efflux while not affecting substrate uptake. Mutation of the putative PIP(2) binding residues R121, K334, and R440 to alanines does not affect NET dimerization but results in decreased substrate efflux that is not altered upon PIP(2) depletion; this indicates that PIP(2) interactions with these residues affect NET-mediated efflux. A dysregulation of norepinephrine and PIP(2) signaling have both been implicated in neuropsychiatric and cardiovascular diseases. This study provides evidence that PIP(2) directly regulates NET organization and function.