Ca²⁺ dependent surface trafficking of norepinephrine transporters depends on threonine 30 and Ca²⁺ calmodulin kinases

The antidepressant-sensitive norepinephrine (NE) transporter (NET) inactivates NE released during central and peripheral neuronal activity by transport into presynaptic cells. Altered NE clearance due to dysfunction of NET has been associated with the development of mental illness and cardiovascular diseases. NET activity in vivo is influenced by stress, neuronal activity, hormones and drugs. We investigated the mechanisms of Ca²⁺ regulation of NET and found that Ca²⁺ influenced both V_max and K_m for NE transport into cortical synaptosomes. Changes in extracellular Ca²⁺ triggered rapid and bidirectional surface trafficking of NET expressed in cultured cells. Deletion of residues 28–47 in the NET NH₂-terminus abolished the Ca²⁺ effect on surface trafficking. Mutagenesis studies identified Thr30 in this region as the essential residue for both Ca²⁺- dependent phosphorylation and trafficking of NET. Depolarization of excitable cells increased surface NET in a Thr30 dependent manner. A proteomic analysis, RNA interference, and pharmacological inhibition supported roles of CaMKI and CaMKII in Ca²⁺-modulated NE transport and NET trafficking. Depolarization of primary noradrenergic neurons in culture with elevated K⁺ increased NET surface expression in a process that required external Ca²⁺ and depended on CaMK activity. Hippocampal NE clearance in vivo was also stimulated by depolarization, and inhibitors of CaMK signaling prevented this stimulation. In summary, Ca²⁺ signaling influenced surface trafficking of NET through a CaMK-dependent mechanism requiring Thr30.