Real-time, spatially resolved analysis of serotonin transporter activity and regulation using the fluorescent substrate, ASP+

Murat Oz, Therissa Libby, Bronwyn Kivell, Vanaja Jaligam, Sammanda Ramamoorthy, Toni S. Shippenberg

Research output: Contribution to journalArticlepeer-review

33 Citations (Scopus)

Abstract

The serotonin transporter (SERT) mediates clearance of serotonin from the synapse, thereby, regulating extracellular serotonin concentrations. Radioligand uptake techniques are typically used to assess SERT function in tissue and heterologous expression systems. The need for sufficient protein in samples, however, requires use of homogenate preparations, potentially masking effects limited to specific cell populations. 4-(4-(dimethylamino)-styryl)-N- methylpyridinium (ASP+) is a fluorescent monoamine transporter substrate that has been used for real-time monitoring of dopamine and norepinephrine transporter function in single cells. The present live cell imaging studies examine the utility of ASP+ for quantifying human SERT function in HEK293 and neuroblastoma cells. We show rapid membrane binding and intracellular ASP+ accumulation in human SERT-expressing cells. Accumulation is saturable; dependent on temperature and the presence of sodium and chloride in the media, and attenuated by serotonin. Acute or prolonged exposure of cells to serotonin re-uptake inhibitors produces a concentration-dependent decrease in accumulation. Similar effects are produced by protein kinase C activation whereas p38 MAPK activation increases ASP + accumulation. These data demonstrate the validity of ASP + as a probe for monitoring SERT function in living cells. Alterations in SERT binding and uptake can be quantified in the same cell and use of a within-cell design permits analysis of time-related alterations in SERT function.

Original languageEnglish
Pages (from-to)1019-1029
Number of pages11
JournalJournal of Neurochemistry
Volume114
Issue number4
DOIs
Publication statusPublished - Aug 2010

Keywords

  • 4-(4-(dimethylamino)-styryl)-N-methylpyridinium
  • live-cell imaging
  • monoamine transporter
  • p38 mitogen-activated protein kinase
  • protein kinase C

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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