title: Ca2+dependent surface trafficking of norepinephrine transporters depends on threonine 30 and Ca2+calmodulin kinases.
abstract: 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 Vmaxand Kmfor 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.
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232-8548, United States. Electronic address: uhna.sung@gmail.com.
authors.forename: Uhna
authors.initials: U
authors.lastname: Sung
authors.affiliation: Institute of Cellular and Integrative Neurosciences, CNRS, Strasbourg, France.
authors.forename: Francesca
authors.initials: F
authors.lastname: Binda
authors.affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232-8548, United States.
authors.forename: Valentina
authors.initials: V
authors.lastname: Savchenko
authors.affiliation: Department of Cellular and Integrative Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, United States.
authors.forename: William A
authors.initials: WA
authors.lastname: Owens
authors.affiliation: Department of Cellular and Integrative Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, United States. Electronic address: daws@uthscsa.edu.
authors.forename: Lynette C
authors.initials: LC
authors.lastname: Daws
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002460:Cell Line; D051379:Mice; D008810:Mice, Inbred C57BL; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013912:Threonine / Q000378:metabolism*
chemical_list: D050484:Norepinephrine Plasma Membrane Transport Proteins; D013912:Threonine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2
keywords: BAPTA/AM (PubChem CID: 2293); BIM (PubChem CID: 2396); Calcium; Calcium calmodulin kinase I; Calcium calmodulin kinase II; Desipramine (PubChem CID: 2995); EGTA (PubChem CID: 6207); KN93 (PubChem CID: 5312122); NE clearance; Norepinephrine transporter; PMSF (PubChem CID: 4784); STO609 (PubChem CID: 16760660); Thapsigargin (PubChem CID: 446378); Trafficking; W7 (PubChem CID: 124887)