Recognition of the muscarinic receptor by its endogenous neurotransmitter: Binding of [³H]acetylcholine and its modulation by transition metal ions and guanine nucleotides

Agonist binding to the muscarinic receptor in rat cerebral cortex membranes was studied by using the neurotransmitter itself, [³H]acetylcholine ([³H]AcCho). By using 10 μM atropine or oxotremorine to define specific binding, it was possible to demonstrate specific binding of [³H]AcCho that was sensitive to muscarinic but not to nicotinic ligands. Equilibrium binding experiments with 5-240 nM [³H]AcCho indicated specific binding of the ligand to a saturable population of muscarinic receptors (361 ± 29 fmol/mg of protein; K(d) = 76 ± 17 nM). This value represented 25% of the available binding sites for a labeled antagonist in the same preparation and corresponds to the proportion of high-affinity agonist binding sites observed previously in competition experiments with labeled antagonists. Inclusion of transition metal ions (e.g., 2 mM Ni²⁺) in the assay increased the equilibrium binding of [³H]AcCho (628 ± 38 fmol/mg of protein, K(d) = 86 ± 21 nM) but did not affect equilibrium binding of ³H-labeled antagonists, indicating conversion of low- into high-affinity muscarinic agonist binding sites. The increase developed slowly over 30 min of incubation at 25°C but could be reversed rapidly (≃2 min) by the chelating agent EDTA or by guanine nucleotides. These data directly reveal a slow though quickly reversible interconversion of low- into high-affinity muscarinic agonist binding sites.