Inhibition of acetylcholine muscarinic M1 receptor function by the M1-selective ligand muscarinic toxin 7 (MT-7)

1 MT-7 (1-30 nM), 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)]-guanosine-5'-O-(3-thio)triphosphat ([S-35]-GTP gamma S) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M-1 receptor subtype. 2 MT-7 failed to affect the ACh-stimulated [S-35]-GTP gamma S binding in membranes of CHO cells expressing either the M-2, M-3, or M-4 receptor subtype. 3 In N1E-115 neuroblastoma cells endogenously expressing the M-1 and M-4 receptor subtypes, MT-7 (0.3-3.0 nM) 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. 4 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 EC50 value. 5 In CHO/M-1 cell membranes, MT-7 (0.05-25 nM) reduced the specific binding of 0.05, 1.0 and 15 nM [H-3]-N-methylscopolamine ([H-3]-NMS) in a concentration-dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT-7 (3 nhl) decreased the dissociation rate of[H-3]-NMS by about 5 fold. 6 CHO/M-1 cell membranes preincubated with MT-7 (10 nM) and washed by centrifugation and resuspension did not recover control [H-3]-NMS binding for at least 8 h at 30 degrees C. 7 It is concluded that MT-7 acts as a selective noncompetitive antagonist of the muscarinic hi, receptors by binding stably to an allosteric site.