Journal
PLOS ONE
Volume 6, Issue 1, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0016519
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Funding
- National Institutes of Health (NIH) [R01-GM18360-39, U01-DA019372, R01-DA029706]
- NSF [GK-12 0742551]
- [GM-07752]
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We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC) by developing sensor cells stably expressing a Ca2+ permeable LGIC and a genetically encoded Forster (or fluorescence) resonance energy transfer (FRET)-based calcium sensor. In particular, we describe separate lines with human alpha 7 and human alpha 4 beta 2 nicotinic acetylcholine receptors, mouse 5-HT3A serotonin receptors and a chimera of human alpha 7/mouse 5-HT3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.
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