A snake toxin against muscarinic acetylcholine receptors: Amino acid sequence, subtype specificity and effect on guinea-pig ileum

Abstract

The sequence of muscarinic toxin 1 (MT1) from Dendroaspis angusticeps (green mamba) was determined (66 amino acids, M r 7509). The central part, peptide 25–40, is rich in hydrophobic amino acids, which is a characteristic of muscarinic toxins. MT1 started to inhibit [ 3H]-NMS ( N-methylscopolamine) binding to synaptosomal membranes of porcine brain (contains all five receptor subtypes) at about 1 nM and to membranes from pig heart muscle (only subtype m2) at about 1 μM. Binding of [ 3H]-AF-DX 384 to heart was inhibited with an ic 50 of 14 μM and to brain in two steps. In the first step (IC 50 = 32 nM) binding decreased by 37%, indicating that the toxin acted on m1 or m4 receptors, each accounting for about 40% of total receptor content. The second step was similar to the effect on heart. Pirenzepine inhibited binding of [ 125I]-MT1 to brain receptors with an ic 50 of 6.5 nM, corresponding to a K i, of about 6 nM. Literature values of K i for pirenzepine are 16–18 nM for m1 and ⩾ 120 mM for other subtypes. This indicates binding to m1 receptors. [ 125I]-MT1 bound to brain with a K d of 20 nM and a Hill coefficient of 1.0 i.e. one toxin molecule per receptor. In guinea-pig ileum, MT1 (670 nM) produced a rapid contraction, reversible by atropine. The toxin may be an agonist, but might also cause contraction by inducing acetylcholine release by a different mechanism.