title: 4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP+) is a fluorescent substrate for the human serotonin transporter.
abstract: Monoamine transporters terminate synaptic neurotransmission and are molecular targets for antidepressants and psychostimulants. Fluorescent reporters can monitor real-time transport and are amenable for high-throughput screening. However, until now, their use has mostly been successful to study the catecholamine transporters but not the serotonin (5HT) transporter. Here, we use fluorescence microscopy, electrophysiology, pharmacology, and molecular modeling to compare fluorescent analogs of 1-methyl-4-phenylpyridinium (MPP(+)) as reporters for the human serotonin transporter (hSERT) in single cells. The fluorescent substrate 4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP(+)) exhibits superior fluorescence uptake in hSERT-expressing HEK293 cells than other MPP(+) analogs tested. APP(+) uptake is Na(+)- and Cl(-)-dependent, displaced by 5HT, and inhibited by fluoxetine, suggesting APP(+) specifically monitors hSERT activity. ASP(+), which was previously used to study catecholamine transporters, is 10 times less potent than APP(+) at inhibiting 5HT uptake and has minimal hSERT-mediated uptake. Furthermore, in hSERT-expressing oocytes voltage-clamped to -60 mV, APP(+) induced fluoxetine-sensitive hSERT-mediated inward currents, indicating APP(+) is a substrate, whereas ASP(+) induced hSERT-mediated outward currents and counteracted 5HT-induced hSERT currents, indicating ASP(+) possesses activity as an inhibitor. Extra-precise ligand receptor docking of APP(+) and ASP(+) in an hSERT homology model showed both ASP(+) and APP(+) docked favorably within the active region; accordingly, comparable concentrations are required to elicit their opposite electrophysiological responses. We conclude APP(+) is better suited than ASP(+) to study hSERT transport fluorometrically.
authors.affiliation: Graduate Training Program in Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee 23235, USA. esolis3@vcu.edu.
authors.forename: Ernesto
authors.initials: E
authors.lastname: Solis
authors.forename: Igor
authors.initials: I
authors.lastname: Zdravkovic
authors.forename: Ian D
authors.initials: ID
authors.lastname: Tomlinson
authors.forename: Sergei Y
authors.initials: SY
authors.lastname: Noskov
authors.forename: Sandra J
authors.initials: SJ
authors.lastname: Rosenthal
authors.forename: Louis J
authors.initials: LJ
authors.lastname: De Felice
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011726:Pyridinium Compounds / Q000737:chemistry / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
chemical_list: C572632:4-(4-(dimethylamino)phenyl)-1-methylpyridinium; D000814:Aniline Compounds; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins