Inhibition of acetylcholine muscarinic M(1) receptor function by the M(1)-selective ligand muscarinic toxin 7 (MT-7).
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)S]-guanosine-5'-O-(3-thio)triphosphate ([(35)S]-GTPgammaS) 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]-GTPgammaS 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 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. 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 nM) reduced the specific binding of 0.05, 1.0 and 15 nM [(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 nM) decreased the dissociation rate of [(3)H]-NMS by about 5 fold. CHO/M(1) cell membranes preincubated with MT-7 (10 nM) and washed by centrifugation and resuspension did not recover control [(3)H]-NMS binding for at least 8 h at 30 degrees 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.
Olianas
M C
MC
Section on Biochemical Pharmacology, Department of Neuroscience, University of Cagliari, via Porcell 4, 09124 Cagliari, Italy.
Maullu
C
C
Adem
A
A
Mulugeta
E
E
Karlsson
E
E
Onali
P
P
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D018678:Cholinergic Agents / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004546:Elapid Venoms / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D018727:Muscarinic Antagonists / Q000494:pharmacology*; D019832:N-Methylscopolamine / Q000494:pharmacology; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic / Q000378:metabolism*; D014316:Tritium
D018678:Cholinergic Agents; D004546:Elapid Venoms; D007295:Inositol Phosphates; D018727:Muscarinic Antagonists; D043643:Receptor, Muscarinic M1; D011976:Receptors, Muscarinic; C419876:muscarinic toxin 7; D014316:Tritium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002217:Carbachol; D000242:Cyclic AMP; D000109:Acetylcholine; D019832:N-Methylscopolamine