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

D. Gurwitz, Y. Kloog, M. Sokolovsky

Research output: Contribution to journalArticlepeer-review

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 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.

Original languageEnglish
Pages (from-to)3650-3654
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume81
Issue number12 I
DOIs
StatePublished - 1984

Fingerprint

Dive into the research topics of 'Recognition of the muscarinic receptor by its endogenous neurotransmitter: Binding of [3H]acetylcholine and its modulation by transition metal ions and guanine nucleotides'. Together they form a unique fingerprint.

Cite this