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

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Abstract
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 .mu.M 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; Kd = 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, Kd = 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.degree. C but could be reversed rapidly (.apprxeq. 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.