Molecular Pharmacology, Vol 20, 492-497, Copyright © 1981 by the American Society for Pharmacology and Experimental Therapeutics

Regeneration of a Functionally Active Rat Brain Muscarinic Receptor by D-Penicillamine after Inhibition with Methylmercury and Mercuric Chloride

Evidence for Essential Sulfhydryl Groups in Muscarinic Receptor Binding Sites

¹ Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201

The molecular mechanism of methylmercury and mercuric chloride inhibition of brain muscarinic acetylcholine receptor is investigated. Both mercuric cations strongly inhibit L-[³H]quinuclidinyl benzilate ([³H]QNB) binding to rat brain lysed synaptosomes. Mercuric chloride is 350 times more potent as an inhibitor of [³H]QNB binding than is methylmercury. Inhibition of the agonist binding site by methylmercury is demonstrated by the competitive action of carbamylcholine chloride on [³H]QNB binding. D-Penicillamine is found to chelate mercuric cations from the receptor binding site and regenerate the [³H]QNB binding in a concentration-dependent manner. The tightness of mercuric chloride interaction with the receptor binding site is demonstrated by measuring [³H] QNB binding before and after extensive washing. The correlation between mercuric chloride inhibition and D-penicillamine regeneration of [³H]QNB binding emphasizes the involvement of sulfhydryl groups in muscarinic receptor binding site. These essential sulfhydryl groups may have a significant role in the proper functional configuration of the receptor binding site. Blocking these essential sulfhydryl groups is suggested to be the molecular mechanism of inhibition of brain muscarinic receptors by these mercurials. Through mercuric chloride inhibition and D-penicillamine regeneration of [³H]QNB binding, the mercuric cation apparently stabilizes or protects the binding site (compared with control) while the protein is subjected to experimental protocol. This may provide a basis for using mercuric chloride as a probe to protect the receptor binding site in solubilization, isolation, and purification of muscarinic receptors.

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ACKNOWLEDGMENTS We are grateful to Dr. M. E. Eldefrawi and Dr. T. Herrmann for their valuable suggestions and critically reviewing the manuscript.

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