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 microM 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 degrees C but could be reversed rapidly (approximately equal to 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.
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent; D002474:Cell-Free System; D002540:Cerebral Cortex / Q000378:metabolism; D003035:Cobalt / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D008345:Manganese / Q000494:pharmacology; D009532:Nickel / Q000494:pharmacology; D009711:Nucleotides / Q000494:pharmacology; D051381:Rats; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*; D013329:Structure-Activity Relationship
D002413:Cations, Divalent; D006150:Guanine Nucleotides; D009711:Nucleotides; D011976:Receptors, Muscarinic; D003035:Cobalt; D008345:Manganese; D009532:Nickel; D000109:Acetylcholine