title: Calcium-dependent interactions of the human norepinephrine transporter with syntaxin 1A.
abstract: The norepinephrine (NE) transporter (NET) terminates noradrenergic signaling by clearing released NE at synapses. The activity of NET can be rapidly regulated by depolarization and receptor activation via Ca2+ and kinase/phosphatase-linked pathways. The SNARE protein syntaxin 1A (SYN1A) interacts with NET and influences transporter surface trafficking and catalytic activity. In this study, we establish a link between changes in intracellular Ca2+ and SYN1A/NET interactions. SYN1A influenced NE transport only in the presence of Ca2+ in brain cortical synaptosomes. Although NET/SYN1A associations were sensitive to manipulations of Ca2+ in CHO cells, in vitro binding experiments using purified NET and SYN1A fusion proteins demonstrated a lack of direct Ca2+ sensitivity. Disruption of NET/SYN1A interaction abolished inhibition of NE transport by phorbol ester (PMA) to activate protein kinase C (PKC), but had no effect on transport inhibition by the Ca2+ calmodulin kinase (CaMK) inhibitor KN93. Furthermore, PMA enhanced Ca2+-dependent modulation of NE transport in synaptosomes. Our data reveal roles for SYN1A in the Ca2+-dependent regulation of NET, likely reliant on regulation by PKC signaling, but independent of CaMK.
authors.affiliation: Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-8548, USA.
authors.forename: Uhna
authors.initials: U
authors.lastname: Sung
authors.forename: Randy D
authors.initials: RD
authors.lastname: Blakely
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D006224:Cricetinae; D003412:Cricetulus; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008954:Models, Biological; D009474:Neurons / Q000378:metabolism; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D050827:Syntaxin 1 / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection / Q000379:methods
chemical_list: D050484:Norepinephrine Plasma Membrane Transport Proteins; D010703:Phorbol Esters; C493227:SLC6A2 protein, human; D050827:Syntaxin 1; D011493:Protein Kinase C; D002118:Calcium