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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 Förster or fluorescence resonance energy transfer FRET-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT3A serotonin receptors and a chimera of human α7-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.



Autor: John G. Yamauchi, Ákos Nemecz, Quoc Thang Nguyen, Arnaud Muller, Lee F. Schroeder, Todd T. Talley, Jon Lindstrom, David Kleinfel

Fuente: http://plos.srce.hr/



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