title: Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors.
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 Ca(2+) 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-HT(3A) serotonin receptors and a chimera of human α7/mouse 5-HT(3A) 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.
authors.affiliation: Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.
authors.forename: John G
authors.initials: JG
authors.lastname: Yamauchi
authors.forename: Ákos
authors.initials: Á
authors.lastname: Nemecz
authors.forename: Quoc Thang
authors.initials: QT
authors.lastname: Nguyen
authors.forename: Arnaud
authors.initials: A
authors.lastname: Muller
authors.forename: Lee F
authors.initials: LF
authors.lastname: Schroeder
authors.forename: Todd T
authors.initials: TT
authors.lastname: Talley
authors.forename: Jon
authors.initials: J
authors.lastname: Lindstrom
authors.forename: David
authors.initials: D
authors.lastname: Kleinfeld
authors.forename: Palmer
authors.initials: P
authors.lastname: Taylor
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000032:analysis*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D015640:Ion Channel Gating; D058446:Ligand-Gated Ion Channels / Q000378:metabolism*; D008024:Ligands; D051379:Mice; D011978:Receptors, Nicotinic / Q000378:metabolism; D011985:Receptors, Serotonin / Q000378:metabolism
chemical_list: D058446:Ligand-Gated Ion Channels; D008024:Ligands; D011978:Receptors, Nicotinic; D011985:Receptors, Serotonin; D002118:Calcium