PURIFICATION AND PROPERTIES OF M1-TOXIN, A SPECIFIC ANTAGONIST OF M1 MUSCARINIC RECEPTORS

The venom of the Eastern green mamba from Africa, Dendroaspis angustic eps, was found to block the binding of H-3-quinuclidinyl benzilate to pure m1 and m4 muscarinic ACh receptors expressed in Chinese hamster o vary cells. The principal toxin in the venom with anti-m1 muscarinic a ctivity was purified by gel filtration and reversed-phase HPLC. This t oxin has 64 amino acids, a molecular mass of 7361 Da, and an isoelectr ic point of 7.04. Its cysteine residues are homologous with those in c urare-mimetic alpha-neurotoxins, and with those in fasciculin, which i nhibits AChE. At low concentrations the toxin blocked m1 receptors ful ly and pseudoirreversibly while having no antagonist activity on m2-m5 receptors; the toxin is therefore named ''m1-toxin.'' At higher conce ntrations m1-toxin interacted reversibly with m4 receptors, and half o f the toxin dissociated in 20 min at 25-degrees-C. The affinity of m1- toxin is therefore much higher for m1 than for m4 receptors. By compar ison with m1-toxin, pirenzepine has sixfold higher affinity for m1 tha n for m4 receptors. Autoradiographs of muscarinic receptors in the rat brain demonstrated that m1-toxin blocked the binding of 2 nm H-3-pire nzepine only in regions known to bind m1-specific antibodies. Thus, m1 -toxin is a much more selective ligand than pirenzepine for functional and binding studies of m1 muscarinic receptors.