Title

M1-Toxin: A Novel Selective Ligand For The M1 Muscarinic Acetylcholine Receptor

Date of Award

1992

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Molecular and Cellular Pharmacology

First Committee Member

Lincoln T. Potter, Committee Chair

Abstract

The venom of the Eastern green mamba, Dendroaspis angusticeps, was found to block the binding of $\sp3$H-N-methylscopolamine (NMS) to pure m1 muscarinic receptors in Chinese hamster ovary (CHO) cells. The most active antimuscarinic component in the venom was purified by gel filtration and reversed-phase HPLC. It was found to have a molecular mass of 7361 D and a unique primary structure. Its cysteine residues are homologous with those in curaremimetic $\alpha$-neurotoxins, and with those in fasciculin, an anticholinesterase toxin. The specificity of the new toxin was tested using CHO cells expressing m1-m5 muscarinic receptors individually. A concentration of 150 ng/ml blocked all m1 receptors. With concentrations sixty-five fold higher, there was partial blockade of m4 receptors, with no effect on m2, m3 or m5 receptors. The purified toxin was therefore named "m1-toxin". As expected, the binding of 1 nM $\sp3$H-pirenzepine to m1 receptors in the hippocampus, cortex and striatum was inhibited. Assays using intact m1 CHO cells demonstrated that the toxin bound to an extracellular site on the m1 receptor. m1-Toxin retained its activity and selectivity in solution. When applied before other ligands, m1-toxin prevented the binding of $\sp3$H-antagonists to m1 receptors, and it blocked the ability of carbachol to increase phosphoinositide turnover in hippocampal slices. When applied after $\sp3$H-NMS, m1-toxin slowed the dissociation of the $\sp3$H-antagonist by a factor of four. This demonstrated simultaneous binding of the toxin and $\sp3$H-NMS, and that antagonists do not block the binding of m1-toxin. The binding of the toxin to m1 receptors was extremely rapid, with inhibition of $\sp3$H-pirenzepine binding occurring within thirty seconds at 4$\sp\circ$C. The effect of m1-toxin was essentially irreversible, with no recovery of $\sp3$H-pirenzepine binding sites for at least eight hours at 25$\sp\circ$C. m1-Toxin was labelled with Na$\sp{125}$I, but lost antimuscarinic activity. Autoradiograms of the binding of $\sp3$H-antagonists in the rat brain in the absence and presence of m1-toxin demonstrated that the toxin blocked the binding of antagonists only in regions known to bind m1-specific antibodies. The selective antagonistic properties of m1-toxin appear excellent for functional, anatomical and binding studies of the m1 muscarinic acetylcholine receptor.

Keywords

Health Sciences, Pharmacology

Link to Full Text

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