Real-time, spatially resolved analysis of serotonin transporter activity and regulation using the fluorescent substrate, ASP+.
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.
Integrative Neuroscience Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, US Department of Health and Human Services, Baltimore, Maryland 21224, USA.
Murat
M
Oz
Therissa
T
Libby
Bronwyn
B
Kivell
Vanaja
V
Jaligam
Sammanda
S
Ramamoorthy
Toni S
TS
Shippenberg
D001667:Binding, Competitive / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes; D006801:Humans; D018715:Microscopy, Video / Q000379:methods; D009474:Neurons / Q000378:metabolism*; D011726:Pyridinium Compounds; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000378:metabolism*; D017367:Selective Serotonin Reuptake Inhibitors; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors
D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D012701:Serotonin; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors; C032315:4-(4-dimethylaminostyryl)-1-methylpyridinium