Solubilization with digitonin alters the kinetics of pirenzepine binding to muscarinic receptors from rat forebrain and heart

The binding properties of the putative M₁ selective antagonist pirenzepine (PZ) to muscarinic acetylcholine receptor (mAChR) embedded in membranes and freed by solubilization with digitonin have been studied in the rat forebrain and heart. In the forebrain membranes, the k₊₁ and k_-1 of [³H]PZ binding was 7.06 x 10⁵ min^-1 M^-1 and 1.84 x 10^-3 min^-1 (K(d) = 3.34 nM), respectively, whereas those in heart membranes were estimated to be 1.21 x 10⁵ min^-1 M^-1 and 7.46 x 10^-3 min^-1 (K(d) = 66 nM) by an indirect method. After solubilization, the kinetic parameters were determined as 3.17 x 10⁵ min^-1 M^-1 and 2.23 x 10^-3 min^-1 (K(d) = 7.16 nM) in the forebrain and 2.41 x 10⁵ min^-1 M^-1 and 1.41 x 10^-3 min^-1 (K(d) = 6.35 nM) in the heart. Similar dissociation constants were obtained from equilibrium saturation binding studies of [³H]PZ. Both high and low affinity binding sites for PZ were found in the forebrain membranes, whereas only one low affinity site was detected in the heart membranes. After solubilization, the inhibition curves in both tissues were better fitted to a one-site model with similar K(i) values. The affinity of the agonist carbachol was decreased greatly in the heart and decreased slightly in the forebrain after solubilization to IC₅₀ values that were similar in both tissues. Although 5'-guanylylimidodiphosphate was albe to reduce the affinity of carbachol for membrane bound mAChR and to increase slightly the binding of (-)-[³H]quinuclidinyl benzilate it had no effect on ligand binding to the solubilized mAChR. The affinities of classic antagonists (-)-[³H]quinuclidinyl benzilate and atropine were not altered significantly by solubilization. Our results suggest that several different factors appear to be involved in the association and dissociation processes of PZ binding to the putative M₁ (in forebrain) and M₂ (in heart) mAChR in membranes. Most of these factors were separated from the receptors by solubilization under our conditions.