m2-toxin: A selective ligand for M2 muscarinic receptors

Selective ligands are needed for distinguishing the functional roles of M2 receptors in tissues containing several muscarinic receptor subtypes. Becau se the venom of the green mamba Dendroaspis angusticeps contains the most s pecific antagonists known for M1 and M4 receptors (m1-toxin and m4-toxin), it was screened for toxins that inhibit the binding of [H-3]N-methylscopola mine ([H-3]NMS) to cloned M2 receptors. Desalted venom had as much anti-M2 as anti-M4 activity. The most active anti-M2 toxin in the venom was isolate d by gel filtration, cation-exchange chromatography, and reversed-phase HPL C, and called m2-toxin. Spectrometry yielded a mass of 7095 Da, and N-termi nal sequencing of 53 amino acids showed RICHSQMSSQPPTTTFCRVNSCYRRTLRDPHDPRG TIIVRGCGCPRMKPGTKL. This sequence is more homologous to antinicotinic than antimuscarinic toxins, but it lacks three almost invariant residues of anti nicotinic toxins required for their activity. m2-Toxin fully blocked the bi nding of [H-3]NMS and [H-3]oxotremorine-M to M2 receptors with Hill coeffic ients near 1, and blocked 77% of the binding sites for 0.1 nM [H-3]NMS in t he rat brainstem (K-i = 11 nM). Concentrations that fully blocked cloned M2 receptors had no effect on M4 receptors, but slightly increased [H-3]NMS b inding to M1 receptors, an allosteric effect. In accord with these results, light microscopic autoradiography of the rat brain showed that m2-toxin de creased [H-3]NMS binding in regions rich in M2 receptors and increased bind ing in regions rich in M1 receptors. Thus m2-toxin is a novel M2-selective, short-chain neurotoxin that may prove useful for binding and functional st udies.