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

Abstract

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.

Figure 1

Effects of MT-7 on ACh stimulation of [³⁵S]-GTPγS binding to membranes of CHO cells expressing the cloned human M₁ to M₄ receptors. The [³⁵S]-GTPγS binding stimulated by ACh was measured in the absence (control) and in the presence of the indicated concentrations of MT-7. Data are the mean±s.e.mean of three experiments.

Figure 2

Effects of MT-7 on CCh-stimulated [³H]-IPs accumulation (A) and CCh-induced inhibition of PACAP38-stimulated cyclic AMP accumulation (B) in N1E-115 neuroblastoma cells. Cells were pretreated with the indicated concentrations of MT-7 for 30 min at 37°C and then exposed to the indicated concentrations of CCh. In (B), PACAP 38 was also added at the final concentrations of 10 nM. Data are the mean±s.e.mean of three experiments for both assays.

Figure 3

Inhibition of specific [³H]-NMS binding to CHO/M₁ cell membranes by MT-7. Binding assays were performed at three concentrations of the radioligand in the presence of the indicated concentrations of MT-7. Data are expressed as per cent binding in the absence of MT-7 and are the mean±s.e.mean of three experiments.

Figure 4

Effects of MT-7 on atropine-induced [³H]-NMS dissociation from M₁ receptors. CHO/M₁ cell membranes were incubated at 30°C first with 1 nM [³H]-NMS to equilibrium for 60 min and then with either vehicle (control) or 3 nM MT-7 for 20 min. The dissociation [³H]-NMS was started by the addition of 10 μM atropine and the incubation was stopped at the indicated time points. [³H]-NMS binding data are expressed as ln B/Be, where B is the amount of [³H]-NMS specifically bound at the indicated time and Be is the specific binding determined at zero time. Lines represent the least-squares linear regressions of the data. Values are the mean of three experiments.

Figure 5

Stable inhibition of [³H]-NMS binding to M₁ receptors by MT-7. CHO/M₁ cell membranes were pretreated with either vehicle (control) or 100 nM MT-7, centrifuged and resuspended in fresh buffer. Aliquots of the membrane suspension were incubated in the presence of 1.5 nM [³H]-NMS at 30°C for the time periods indicated in abscissa. Data are the mean±s.e.mean of three experiments.