A snake toxin against muscarinic acetylcholine receptors: amino acid sequence, subtype specificity and effect on guinea-pig ileum.
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 microM. Binding of [3H]-AF-DX 384 to heart was inhibited with an IC50 of 14 microM and to brain in two steps. In the first step (IC50 = 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 IC50 of 6.5 nM, corresponding to a Ki of about 6 nM. Literature values of Ki for pirenzepine are 16-18 nM for m1 and > or = 120 mM for other subtypes. This indicates binding to m1 receptors. mM for other subtypes. This indicates binding to m1 receptors. [125I]-MT1 bound to brain with a Kd 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.
Jolkkonen
M
M
Department of Biochemistry, Biomedical Centre, Uppsala, Sweden.
Adem
A
A
Hellman
U
U
Wernstedt
C
C
Karlsson
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D004546:Elapid Venoms / Q000737:chemistry / Q000633:toxicity*; D005612:Freeze Drying; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D008024:Ligands; D008969:Molecular Sequence Data; D018727:Muscarinic Antagonists / Q000737:chemistry / Q000633:toxicity*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000294:innervation; D009206:Myocardium / Q000378:metabolism; D010890:Pirenzepine / Q000378:metabolism; D011869:Radioligand Assay; D011976:Receptors, Muscarinic / Q000187:drug effects*; D013552:Swine; D013570:Synaptic Membranes / Q000187:drug effects / Q000378:metabolism; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism
D004546:Elapid Venoms; D007457:Iodine Radioisotopes; D008024:Ligands; D018727:Muscarinic Antagonists; D011976:Receptors, Muscarinic; D010890:Pirenzepine; D001285:Atropine