Molecular Pharmacology, Vol 14, 575-586, Copyright © 1978 by the American Society for Pharmacology and Experimental Therapeutics

Influence of Sulfhydryl Reagents and Heavy Metals on the Functional State of the Muscarinic Acetylcholine Receptor in Rat Brain

¹ Center for Brain Research, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Membranes from rat brain contain several groups that react with sulfhydryl reagents to influence muscarinic acetylcholine receptor binding. p-Chloromercuribenzoate (PCMB) reacts with a group(s) within or under the allosteric control of the receptor binding site to inhibit both agonist and antagonist binding. Receptors can be protected from PCMB inactivation by the presence of receptor ligands (agonists or antagonists), and the inactivation can be reversed by subsequent treatment with organic sulfhydryls. Reductive alkylation of neural membranes with N-ethylmaleimide (NEM) can prevent much of the inhibition of antagonist, but not agonist, binding by subsequent PCMB treatment, suggesting that it is the presence of a mercuribenzoate, but not an ethylmaleimide residue, within the binding site that is inimical to receptor binding. NEM treatment increases agonist binding by converting receptors from a state of low agonist affinity to high agonist affinity, whereas both states have the same high affinity for receptor antagonists. The presence of agonists but not antagonists during the NEM treatment enhances the ability of NEM to increase agonist affinity. Prior treatment with low concentrations of PCMB abolishes the ability of NEM subsequently to increase agonist binding, even when binding site-saturating concentrations of receptor ligands are included during the PCMB treatment to protect the binding site—a finding which suggests that NEM exerts its influence on agonist binding through interaction with a group contiguous to the receptor binding site. Transition metal ions, which appear to interact with all of the NEM- and PCMB-reactive moieties, increase agonist binding, decrease agonist binding, or decrease both agonist and antagonist binding, depending on the concentration of metal. Membranes from various areas of the brain contain receptors that differ in their distribution between the high- and low-agonist-affinity forms; in terms of the affinity of muscarinic receptors for agonists, brain stem>>telencephalon>hippocampus. Receptors from different brain areas also display different sensitivities to sulfhydryl reagents and heavy metals.

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