Molecular distinction between calf heart and brain muscarine receptors: different N-ethylmaleimide modulation of agonist binding.

Muscarinic acetylcholine receptors in calf heart and forebrain membranes were identified by binding of 1-[benzilic-4,4'-3H]quinuclidinyl benzilate ([3H]QNB). We were able to solubilise these receptors with a yield of 50% of the proteins by treatment of the membranes with digitonin. The existence of two or more receptor subclasses with different agonist affinity in the heart membranes was evidenced by the shallow carbachol/[3H]QNB competition binding curves. The receptors displayed only low agonist affinity, in the presence of GTP as well as after solubilisation. The alkylating reagent N-ethylmaleimide (NEM) caused a 70-fold increase in agonist affinity for the solubilised receptors whereas GTP was ineffective. A similar difference in affinity was observed for the membranes when agonist competition curves in the presence of NEM were compared to those in the presence of GTP. NEM caused only a 2- to 3-fold increase of the agonist affinity for solubilised brain cortex membranes. These data suggest that heart and brain muscarine receptors are structurally different.