Inhibition of acetylcholine muscarinic M 1 receptor function by the M 1 ‐selective ligand muscarinic toxin 7 (MT‐7)

MT‐7 (1–30 n M ), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M 1 receptor, inhibited the acetylcholine (ACh)‐stimulated [ 35 S]‐guanosine‐5′‐O‐(3‐thio)triphosphate ([ 35 S]‐GTPγS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M 1 receptor subtype. MT‐7 failed to affect the ACh‐stimulated [ 35 S]‐GTPγS binding in membranes of CHO cells expressing either the M 2 , M 3 or M 4 receptor subtype. In N1E‐115 neuroblastoma cells endogenously expressing the M 1 and M 4 receptor subtypes, MT‐7 (0.3–3.0 n M ) inhibited the carbachol (CCh)‐stimulated inositol phosphates accumulation, but failed to affect the CCh‐induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38‐stimulated cyclic AMP accumulation. In both CHO/M 1 and N1E‐115 cells the MT‐7 inhibition consisted in a decrease of the maximal agonist effect with minimal changes in the agonist EC 50 value. In CHO/M 1 cell membranes, MT‐7 (0.05–25 n M ) reduced the specific binding of 0.05, 1.0 and 15 n M [ 3 H]‐N‐methylscopolamine ([ 3 H]‐NMS) in a concentration‐dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT‐7 (3 n M ) decreased the dissociation rate of [ 3 H]‐NMS by about 5 fold. CHO/M 1 cell membranes preincubated with MT‐7 (10 n M ) and washed by centrifugation and resuspension did not recover control [ 3 H]‐NMS binding for at least 8 h at 30°C. It is concluded that MT‐7 acts as a selective noncompetitive antagonist of the muscarinic M 1 receptors by binding stably to an allosteric site.British Journal of Pharmacology (2000) 131 , 447–452; doi: 10.1038/sj.bjp.0703606