ACUTE REGULATION OF NOREPINEPHRINE TRANSPORT - II - PKC-MODULATED SURFACE EXPRESSION OF HUMAN NOREPINEPHRINE TRANSPORTER PROTEINS

Norepinephrine (NE) transporters (NETs) found in the neuronal plasma m embrane mediate the removal of NE from the extracellular space, limiti ng the activation of adrenoceptors at noradrenergic synapses. Our prev ious studies with the noradrenergic neuroblastoma SK-N-SH have reveale d a muscarinic receptor-triggered regulation of NET surface density an d transport capacity, mediated in part by protein kinase C activation. Low abundance of NET proteins in this native cell model, however, pre clude direct confirmation of altered trafficking of NET proteins. In o ur study, we monitored the activity and surface distribution of human NET proteins in transient and stably-transfected cell lines after appl ication of kinase activators and inhibitors. Using hNET stably transfe cted HEK-293 and LLC-PK1 cells, as well as transiently transfected COS -7 cells, we demonstrate that PKC-activating phorbol esters, beta-PMA or beta-PDBu selectively diminish I-NE transport capacity (Vmax) with little change in NE K-m. Effects of phorbol esters are rapid, stereosp ecific and blocked by protein kinase C inhibitors, staurosporine and b isindolylmaleimide I. As in SK-N-SH cells, beta-PMA induces a reductio n in intact cell [H-3]nisoxetine binding sites with no change in nisox etine K-d or total membrane NET density. Cell-surface biotinylation an d confocal immunofluorescence techniques confirm that protein kinase C -dependent reductions in NE transport capacity and whole-cell antagoni st binding density are accompanied by reductions in cell-surface human NET protein expression. Together these findings argue for kinase-modu lated protein trafficking as a potential route for acute regulation of antidepressant-sensitive NE clearance.