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

Authors: Oz, Murat; Libby, Therissa; Kivell, Bronwyn; Jaligam, Vanaja¹; Ramamoorthy, Sammanda²; Shippenberg, Toni S.¹

Source: Journal of Neurochemistry, Volume 114, Number 4, August 2010 , pp. 1019-1029(11)

Publisher: Wiley-Blackwell

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Abstract:

J. Neurochem. (2010) 114, 1019-1029. 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.

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

Document Type: Research article

DOI: http://dx.doi.org/10.1111/j.1471-4159.2010.06828.x

Affiliations: 1: Integrative Neuroscience Section, Intramural Research Program, National Institute On Drug Abuse, National Institutes of Health, U.S. Department of Health and Human Services, Baltimore, Maryland, USA 2: Department of Neurosciences, Medical College of South Carolina, Charleston, South Carolina, USA

Publication date: 2010-08-01