Abstract
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.
Highlights
Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels (LGIC) belonging to the sub-family of Cys-loop receptors
We report on a cell-based assay incorporating the use of Nicotinic acetylcholine receptors (nAChRs) cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) to identify and characterize nAChR agonists and antagonists with a low cost, medium throughput fluorescence assay
The 5-HT3A serotonergic LGIC receptor exists in its simplest form as a homopentamer similar to the a7-nAChR [25]
Summary
Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels (LGIC) belonging to the sub-family of Cys-loop receptors. They are found in the central and peripheral nervous systems of both vertebrate and invertebrate species. Specific nAChR subtypes are recognized pharmaceutical targets for many central nervous system (CNS) diseases and conditions [1]. A particular challenge in therapeutic considerations is achieving subtype selectivity among the nAChRs. Sorting out the desirable actions within the subunit diversity of the nAChR family from the largely unwanted responses, via off-target receptors, is an important facet of the therapeutic approach [9]
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