Abstract

Norepinephrine (NE) transporters (NETs) are high-affinity transport proteins that mediate the synaptic clearance of NE after vesicular release. NETs represent a major therapeutic target for antidepressants and are targets of multiple psychostimulants including amphetamine (AMPH) and cocaine. Recently, we demonstrated that syntaxin 1A (SYN1A) regulates NET surface expression and, through binding to the transporter's NH₂ terminus, regulates transporter catalytic function. AMPH induces NE efflux and may also regulate transporter trafficking. We monitored NET distribution and function in catecholaminergic cell lines (CAD) stably transfected with either full-length human NET (CAD-hNET) or with an hNET N-terminal deletion (CAD-hNETΔ_28-47 cells). In hNET-CAD cells, AMPH causes a slow and small reduction of surface hNET with a modest increase in hNET/SYN1A associations at the plasma membrane. In contrast, in CAD-hNETΔ_28-47 cells, AMPH induces a rapid and substantial reduction in surface hNETΔ_28-47 accompanied by a large increase in plasma membrane hNETΔ_28-47/SYN1A complexes. We also found that AMPH in CAD-hNETΔ_28-47 cells induces a robust increase in cytosolic Ca²⁺ and concomitant activation of calcium/calmodulin-dependent protein kinase II (CaMKII). Inhibition of either the increase in intracellular Ca²⁺ or CaMKII activity blocks AMPH-stimulated hNETΔ_28-47 trafficking and the formation of hNETΔ_28-47/SYN1A complexes. Here, we demonstrate that AMPH stimulation of CAMKII stabilizes an hNET/SYN1A complex. This hNET/SYN1A complex rapidly redistributes, upon AMPH treatment, when mechanisms supported by the transporter's NH₂ terminus are eliminated.