RAPID ASSESSMENT OF LIGAND ACTIONS WITH NICOTINIC ACETYLCHOLINE-RECEPTORS USING CALCIUM DYNAMICS AND FLIPR

Due to the complex mechanism of cation transport by neuronal nicotinic acetylcholine receptors (nAChR), potential ligands of these receptors must be characterized with respect to functional activity and corresp onding subtype selectivity. Conventional radioactive binding assays ha ve been established in high throughput screening (HTS) formats for rap id assessment of ligand subtype selectivity; however, the current radi ometric methods of functionally profiling these ligands are not suitab le for HTS formats. In this article, a high throughput, cell-based ass ay is described wh ch exploits the Ca2+ transport activity of nAChRs a s a method of functional assessment. By coupling the use of Fluo-3, a Ca2+-chelating fluorescent dye, with a fluorescence imaging plate read er (FLIPR), the movement of Ca2+ by nAChRs can be observed. Fluorescen ce changes can be measured simultaneously over an entire 96-well plate , allowing six to eight concentrations of six ligands to be examined i n duplicate on each plate within minutes. In the data presented, 36 nA ChR ligands were functionally profiled in IMR-32 cells, a model of gan glionic nAChR subtypes. The calculated potency values(pEC(50) values) from this fluorescence assay compare well with those determined by Rb- 86(+)-efflux and demonstrate a correlation coefficient of 0.91 between the two methods. Thus, the functional profile of novel nAChR ligands can rapidly be assessed by monitoring the flux of calcium upon activat ion of these ligand gated ion channels (LGICs). Moreover, this HTS ass ay can be easily adapted for any cell line expressing receptors involv ed directly or indirectly with the influx of calcium into the cytosol, including similar LGICs and G-protein coupled receptors. (C) 1998 Wil ey-Liss, Inc.