TY - JOUR
T1 - Real-time, spatially resolved analysis of serotonin transporter activity and regulation using the fluorescent substrate, ASP+
AU - Oz, Murat
AU - Libby, Therissa
AU - Kivell, Bronwyn
AU - Jaligam, Vanaja
AU - Ramamoorthy, Sammanda
AU - Shippenberg, Toni S.
PY - 2010/8
Y1 - 2010/8
N2 - 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.
AB - 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.
KW - 4-(4-(dimethylamino)-styryl)-N-methylpyridinium
KW - live-cell imaging
KW - monoamine transporter
KW - p38 mitogen-activated protein kinase
KW - protein kinase C
UR - http://www.scopus.com/inward/record.url?scp=77954717291&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77954717291&partnerID=8YFLogxK
U2 - 10.1111/j.1471-4159.2010.06828.x
DO - 10.1111/j.1471-4159.2010.06828.x
M3 - Article
C2 - 20524964
AN - SCOPUS:77954717291
SN - 0022-3042
VL - 114
SP - 1019
EP - 1029
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
IS - 4
ER -