Rapid assessment of ligand actions with nicotinic acetylcholine receptors using calcium dynamics and FLIPR

Abstract

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 corresponding subtype selectivity. Conventional radioactive binding assays have been established in high throughput screening (HTS) formats for rapid assessment of ligand subtype selectivity; however, the current radiometric methods of functionally profiling these ligands are not suitable for HTS formats. In this article, a high throughput, cell-based assay is described which exploits the Ca2+ transport activity of nAChRs as a method of functional assessment. By coupling the use of Fluo-3, a Ca2+-chelating fluorescent dye, with a fluorescence imaging plate reader (FLIPR), the movement of Ca2+ by nAChRs can be observed. Fluorescence changes can be measured simultaneously over an entire 96-well plate, allowing six to eight concentrations of six ligands to be examined in duplicate on each plate within minutes. In the data presented, 36 nAChR ligands were functionally profiled in IMR-32 cells, a model of ganglionic nAChR subtypes. The calculated potency values (pEC50 values) from this fluorescence assay compare well with those determined by 86Rb+-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 activation of these ligand gated ion channels (LGICs). Moreover, this HTS assay can be easily adapted for any cell line expressing receptors involved directly or indirectly with the influx of calcium into the cytosol, including similar LGICs and G-protein coupled receptors. Drug Dev. Res. 44:14–20, 1998. © 1998 Wiley-Liss, Inc.