Abstract
The dopamine transporter is a neuronal protein that drives the presynaptic reuptake of dopamine (DA) and is the major determinant of transmitter availability in the brain. Dopamine transporter function is regulated by protein kinase C (PKC) and other signaling pathways through mechanisms that are complex and poorly understood. Here we investigate the role of Ser-7 phosphorylation and Cys-580 palmitoylation in mediating steady-state transport kinetics and PKC-stimulated transport down-regulation. Using both mutational and pharmacological approaches, we demonstrate that these post-translational modifications are reciprocally regulated, leading to transporter populations that display high phosphorylation-low palmitoylation or low phosphorylation-high palmitoylation. The balance between the modifications dictates transport capacity, as conditions that promote high phosphorylation or low palmitoylation reduce transport Vmax and enhance PKC-stimulated down-regulation, whereas conditions that promote low phosphorylation or high palmitoylation increase transport Vmax and suppress PKC-stimulated down-regulation. Transitions between these functional states occur when endocytosis is blocked or undetectable, indicating that the modifications kinetically regulate the velocity of surface transporters. These findings reveal a novel mechanism for control of DA reuptake that may represent a point of dysregulation in DA imbalance disorders.
Highlights
The dopamine transporter establishes synaptic transmitter levels and strength of dopamine neurotransmission
C580A dopamine transporter (DAT) Displays Enhanced Phosphorylation on Ser-7— To determine if phosphorylation of DAT is affected by its level of palmitoylation, we examined the 32P labeling of C580A DAT, which shows an ϳ50% reduction in palmitoylation, and C522A
32P labeling of C580A DAT was enhanced in both basal (263 Ϯ 25% of WT basal, p Ͻ 0.05) and PMA-stimulated conditions (1154 Ϯ 129% of WT basal, p Ͻ 0.001 versus C580A basal; p Ͻ 0.01 versus WT PMA), indicating that lack of palmitoylation on Cys-580 results in elevated basal and stimulated phosphorylation
Summary
The dopamine transporter establishes synaptic transmitter levels and strength of dopamine neurotransmission. Transitions between these functional states occur when endocytosis is blocked or undetectable, indicating that the modifications kinetically regulate the velocity of surface transporters These findings reveal a novel mechanism for control of DA reuptake that may represent a point of dysregulation in DA imbalance disorders. We investigate the relationship between Ser-7 phosphorylation and Cys-580 palmitoylation of DAT in steady-state transport kinetics and PKC-stimulated down-regulation, finding that these modifications are reciprocally regulated and induce opposing effects on Vmax and PKC responsiveness. The functional differences are driven by a trafficking-independent process in which reduced transport correlates with Ser-7 phosphorylation and increased transport correlates with Cys-580 palmitoylation This demonstrates a previously unknown mechanism for control of DAT function and suggests the potential for these modifications to represent sites of dysregulation in DA imbalance disorders
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