Characterizing ligand-gated ion channel receptors with genetically encoded Ca++ sensors

Abstract

We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligandgated 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 α 4β2 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. Concentrationresponse 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 ligandgated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters. © 2011 Yamauchi et al.