Muscarinic Toxins from the Black Mamba Dendroaspis polylepis

Three new toxins acting on muscarinic receptors were isolated from the venom of the black mamba Dendroaspis polylepis. They were called muscarinic toxins α, β, and γ (MTα, MTβ, and MTγ), All of the toxins have four disulphide bonds and 65 or 66 amino acids. The sequences of MTα and MTβ were determined. The muscarinic toxins, of which about 12 have been isolated from venoms of green and black mambas, have 60–98% sequence identity with each other, and are similar to many (about 180) other snake venom components, such as α‐neurotoxins, cardiotoxins, and fasciculins. In contrast to the α‐neurotoxins, muscarinic toxins do not bind to nicotinic acetylcholine receptors. The binding constants of MTα and MTβ were determined for human muscarinic receptors of subtypes m1–m5 stably expressed in Chinese hamster ovary cells. The toxins are less selective than the earlier discovered muscarinic toxins from the green mamba Dendroaspis angusticeps. MTα and the muscarinic toxin MT4 from D. angusticeps differ only in a region of three amino acids (residues 31–33), which are Leu‐Asn‐His in MTα and Ile‐Val‐Pro in MT4. This difference causes a pronounced shift in subtype selectivity. MTα has high affinity to all subtypes, with K i (inhibition constant) values of 23 nM (m1; pK i = 7.64±0.10), 44 nM (m2; p K i = 7.36±0.06), 3 nM (m3: p K i = 8.46±0.14), 5 nM (m4; p K i = 8.32±0.07), and 8 nM (m5; p K i = 8.09±0.07). MT4 has high affinity only to m1 ( K i = 62 nM) and m4 (87 nM) receptors, and low ( K i >1 μM) affinity to m2, m3, and m5. The region at positions 31–33 evidently plays an important role in the toxin‐receptor interaction. MTβ has low affinity for ml and m2 receptors K i >1 μM) and intermediate affinity for m3 (140 nM; p K i = 6.85±0.03), m4 (120 nM; p K i = 6.90±0.06), and m5 (350 nM; p K i = 6.46±0.01). The low affinity of MTβ may reflect a tendency for spontaneous inactivation.