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 M₁ receptor, inhibited the acetylcholine (ACh)-stimulated [³⁵S]-guanosine-5'-O-(3-thio)triphosphate ([³⁵S]-GTPγS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M₁ receptor subtype. 2 MT-7 failed to affect the ACh-stimulated [³⁵S]-GTPγS binding in membranes of CHO cells expressing either the M₂, M₃ or M₄ receptor subtype. 3 In N1E-115 neuroblastoma cells endogenously expressing the M₁ and M₄ 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/M₁ 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/M₁ cell membranes, MT-7 (0.05-25 nM) reduced the specific binding of 0.05, 1.0 and 15 nm [³H]-N-methylscopolamine ([³H]-NMS) in a concentration-dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT-7 (3 nM) decreased the dissociation rate of [³H]-NMS by about 5 fold. 6 CHO/M₁ cell membranes preincubated with MT-7 (10 nM) and washed by centrifugation and resuspension did not recover control [³H]-NMS binding for at least 8 h at 30°C. 7 It is concluded that MT-7 acts as a selective noncompetitive antagonist of the muscarinic M₁ receptors by binding stably to an allosteric site.