title: Single molecule analysis of serotonin transporter regulation using antagonist-conjugated quantum dots reveals restricted, p38 MAPK-dependent mobilization underlying uptake activation.
abstract: The presynaptic serotonin (5-HT) transporter (SERT) is targeted by widely prescribed antidepressant medications. Altered SERT expression or regulation has been implicated in multiple neuropsychiatric disorders, including anxiety, depression and autism. Here, we implement a generalizable strategy that exploits antagonist-conjugated quantum dots (Qdots) to monitor, for the first time, single SERT proteins on the surface of serotonergic cells. We document two pools of SERT proteins defined by lateral mobility, one that exhibits relatively free diffusion, and a second, localized to cholesterol and GM1 ganglioside-enriched microdomains, that displays restricted mobility. Receptor-linked signaling pathways that enhance SERT activity mobilize transporters that, nonetheless, remain confined to membrane microdomains. Mobilization of transporters arises from a p38 MAPK-dependent untethering of the SERT C terminus from the juxtamembrane actin cytoskeleton. Our studies establish the utility of ligand-conjugated Qdots for analysis of the behavior of single membrane proteins and reveal a physical basis for signaling-mediated SERT regulation.
authors: Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37240, USA.
authors: Jerry C
authors: JC
authors: Chang
authors: Ian D
authors: ID
authors: Tomlinson
authors: Michael R
authors: MR
authors: Warnement
authors: Alessandro
authors: A
authors: Ustione
authors: Ana M D
authors: AM
authors: Carneiro
authors: David W
authors: DW
authors: Piston
authors: Randy D
authors: RD
authors: Blakely
authors: Sandra J
authors: SJ
authors: Rosenthal
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002461:Cell Line, Transformed; D002772:Cholera Toxin / Q000494:pharmacology*; D002784:Cholesterol / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016537:Gangliosidosis, GM1 / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D008024:Ligands; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016011:Normal Distribution; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D045663:Quantum Dots; D051381:Rats; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology; D047392:beta-Cyclodextrins / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
chemical_list: D000199:Actins; D004791:Enzyme Inhibitors; D007093:Imidazoles; D008024:Ligands; D019384:Nucleic Acid Synthesis Inhibitors; D011725:Pyridines; D050486:Serotonin Plasma Membrane Transport Proteins; D013873:Thionucleotides; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C083763:8-bromoguanosino-3',5'-cyclic monophosphorothioate; D015638:Cytochalasin D; D012701:Serotonin; D002772:Cholera Toxin; D002784:Cholesterol; D048051:p38 Mitogen-Activated Protein Kinases; D006152:Cyclic GMP; C093642:SB 203580