A rapid microtechnique for the estimation of muscarinic and nicotinic receptor binding parameters using 96-well filtration plates

A microtechnique for the assay of muscarinic and nicotinic receptor binding parameters employing a 96-well filtration multiscreen system has been developed. Utilizing rat cerebral cortex as the receptor source, saturation analysis of [³H]NMS binding revealed a mean B_max and K_d value of 1.95 pmol/mg protein and 0.71 nM, respectively. In competition studies with atropine a K_i value of 1.67 nM was determined. Nicotinic receptor binding studies were also performed with this technique with [³H]Cytisine. The binding constants for both ligands correlated well with established literature values; however, in the nicotinic assays concentrations of ligand at 20 times the K_d were required to develop adequate number of counts. Compared to presently available receptor binding techniques, this newly developed system for muscarinic and nicotinic binding is very simple and convenient to use and offers the advantages of speed, accuracy and reproducibility. In addition, a large number of samples can be assayed. This report also presents the first use of the 96-well filtration multiscreen system for the estimation of muscarinic and nicotinic receptor binding constants. This methodology should prove to be useful for determining the binding characteristics of potential cholinergic ligands.