Analysis of ligand binding to receptors by competition kinetics. Application to muscarinic antagonists in rat brain cortex.

We propose a method for obtaining kinetic parameters of unlabeled ligands which is based on analyzing the effect of their competition on the binding kinetics of a labeled ligand. In this method (competition kinetics), the binding kinetics of a labeled ligand are measured in the absence and in the presence of given concentrations of a competing unlabeled ligand. The rate equations appropriate to the particular kinetic model may be solved employing linear homogeneous differential equations. The rate constants appearing in the final integrated expressions can be evaluated by nonlinear regression. We have demonstrated the validity and applicability of the method to study receptor systems, using the muscarinic receptors in rat brain cortex as an experimental system. We have determined the various kinetic parameters of two muscarinic antagonists (N-methyl-4-[3H]piperidyl benzilate and (-)-N-[3H]methylscopolamine) by following directly their binding kinetics. These parameters were compared with those obtained for the same unlabeled ligands in homogeneous and heterogeneous competition studies. In all cases, the parameters obtained by the competition kinetics method were close to those extracted from direct binding kinetics, and in cases where both types of studies were performed on the same preparations, the parameters obtained by the two methods were essentially identical.