The binding of pirenzepine to digitonin‐solubilized muscarinic acetylcholine receptors from the rat myocardium

Abstract

1 The binding of pirenzepine to digitonin-solubilized rat myocardial muscarinic acetylcholine receptors has been examined at 4°C. 2 Solubilization produced only small changes in the binding of N-methylscopolamine and atropine. In contrast to the low affinity binding of pirenzepine found to be present in the membranes, high affinity binding was detected in the soluble preparation. In both preparations, pirenzepine binding was complex. 3 High affinity pirenzepine binding (K_D ∼ 3 × 10⁻⁸M) to the soluble myocardial receptors could be monitored directly using [³H]-pirenzepine. 4 [³H]-pirenzepine-labelled soluble myocardial receptors have a sedimentation coefficient of 11.1 s. This indicates that [³H]-pirenzepine binds predominantly to the uncoupled form of the receptor. However, [³H]- pirenzepine-agonist competition experiments indicated that the high affinity pirenzepine binding sites are capable of coupling with a guanosine 5′-triphosphate (GTP)-binding protein. Pirenzepine affinities for the soluble myocardial receptors were unaffected by their state of association with the GTP-binding proteins found in the heart. 5 The equilibrium binding properties of the soluble cortical and myocardial receptors were very similar. However, the binding kinetics of the myocardial receptor were much slower. 6 It appears that the membrane environment can affect the affinity of pirenzepine for the rat myocardial muscarinic receptor. Removal of the constraint by solubilization allows the expression of high affinity pirenzepine binding.