BINDING OF MUSCARINIC TOXINS MTX1 AND MTX2 FROM THE VENOM OF THE GREEN MAMBA DENDROASPIS-ANGUSTICEPS TO CLONED HUMAN MUSCARINIC CHOLINOCEPTORS

Muscarinic toxins MTx1 and MTx2 are 7500 mol. wt polypeptides isolated from the venom of the green mamba snake Dendroaspis angusticeps. Prev ious competition binding studies indicate that the MTxs may be selecti ve for the M(1) subtype of muscarinic acetylcholine receptors, The pre sent work was undertaken in order to clarify the muscarinic subtype sp ecificity and functional effects of MTx1 and MTx2. Binding interaction s were determined using H-3-N-methyl scopolamine (NMS) and cloned huma n muscarinic receptor subtypes m1, m2, m3 and m4. Some preliminary fun ctional studies were performed on rabbit vas deferens preparations, wh ich contain M(1) cholinoceptors. MTx1 and MTx2 inhibited H-3-NMS bindi ng to m1 and m3 receptors, with little effect on binding to m2 and m4 receptors. Affinity was higher for m1 receptors: K-i for MTx1 were 48 nM at m1 receptors and 72 nM at m3 receptors, and K-i for MTx2 were 36 4 nM at ml and 1.2 mu M at m3 receptors, At m1 receptors, about 90% of the binding of MTx1 and MTx2 appears to be irreversible. On rabbit va s deferens preparations, MTx1 and MTx2 at concentrations above 50 nM b ehaved in a similar way to the relatively selective M(1)-agonists M(c) N-A-343 and CPCP enyl)carbamoyloxy]-4-pent-2-ynyl-trimethylammonium io dide) by reducing responses to nerve stimulation. The results confirm that MTx1 and MTx2 bind to m1 receptors rather than to m2 or m4 recept ors, but they also reveal a slightly weaker effect at m3 receptors. Th e interaction at mi receptors apears to be essentially irreversible, i mplying that the toxins could be useful tools in studies of the functi onal role of m1 muscarinic receptors.