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 musc arinic toxins alpha, beta, and gamma (MT alpha, MT beta, and MT gamma) . All of the toxins have four disulphide bonds and 65 or 66 amino acid s. The sequences of MT alpha and MT beta were determined. The muscarin ic toxins, of which about 12 have been isolated from venoms of green a nd black mambas, have 60-98% sequence identity with each other, and ar e similar to many (about 180) other snake venom components, such as al pha-neurotoxins, cardiotoxins, and fasciculins. In contrast to the a-n eurotoxins, muscarinic toxins do not bind to nicotinic acetylcholine r eceptors. The binding constants of MT alpha and MT beta were determine d for human muscarinic receptors of subtypes m1-m5 stably expressed in Chinese hamster ovary cells. The toxins are less selective than the e arlier discovered muscarinic toxins from the green mamba Dendroaspis a ngusticeps. MT alpha and the muscarinic toxin MT4 from D. angusticeps differ only in a region of three amino acids (residues 31-33), which a re Leu-Asn-His in MT alpha and Ile-Val-Pro in MT4. This difference cau ses a pronounced shift in subtype selectivity. MT alpha has high affin ity to all subtypes, with K-i (inhibition constant) values of 23 nM (m 1; pK(i) = 7.64 +/- 0.10), 44 nM (m2; pK(i) = 7.36 +/- 0.06), 3 nM (m3 ; pK(i) = 8.46 +/- 0.14), 5 nM (m4; pK(i) = 8.32 +/- 0.07), and 8 nM ( m5; pK(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 mu M) affinity to m2, m3, and m5. The region at positions 31-33 evidently plays an important role in the toxin-receptor interaction. MT beta has low affinity for m1 and m2 receptors (K-i > 1 mu M) and intermediate affinity for m3 (1 40 nM; pK(i) = 6.85 +/- 0.03), m4 (120 nM; pK(i) = 6.90 +/- 0.06), and m5 (350 nM; pK(i) = 6.46 +/- 0.01). The low affinity of MT beta may r eflect a tendency for spontaneous inactivation.