Mikael Jolkkonen

A toxin from the green mamba Dendroaspis angusticeps: Amino acid sequence and selectivity for muscarinic m4 receptors

Muscarinic toxin 3 (MT3) (65 amino acids, four disulphides, M r 7379) was isolated from the venom of the African snake Dendroaspis angusticeps (green mamba) and its amino acid sequence determined. Its ability to inhibit the binding of [3H]N‐methylscopolamine ([3H]NMS) to Chinese hamster ovary cells stably expressing subtypes of muscarinic receptors was studied. MT3 displayed high affinity for the m4 receptor (pK i = 8.7 ± 0.06), 40‐fold lower affinity at m1 receptors (pK i = 7.11 ± 0.17) whereas no inhibition of [3H]NMS binding to m2, m3 and m5 receptors was observed at concentrations up to 1 μM. This makes MT3 the most selective m4 receptor ligand known to date.

Snake toxins with high selectivity for subtypes of muscarinic acetylcholine receptors.

There are five subtypes of muscarinic acetylcholine receptors (M(1) to M(5)) which control a large number of physiological processes, such as the function of heart and smooth muscles, glandular secretion, release of neurotransmitters, gene expression and cognitive functions as learning and memory. A selective ligand is very useful for studying the function of a subtype in presence of other subtypes, which is the most common situation, since a cell or an organ usually has several subtypes. There are many non-selective muscarinic ligands, but only few selective ones. Mambas, African snakes of genus Dendroaspis have toxins, muscarinic toxins, that are selective for M(1), M(2) and M(4) receptors. They consist of 63-66 amino acids and four disulfides which form four loops. They are members of a large group of snake toxins, three-finger toxins; three loops are extended like the middle fingers of a hand and the disulfides and the shortest loop are in the palm of the hand. Some of the toxins target the allosteric site which is located in a cleft of the receptor molecule close to its extracellular part. A possible explanation to the good selectivity is that the toxins bind to the allosteric site, but because of their size they probably also bind to extracellular parts of the receptors which are rather different in the various subtypes. Some other allosteric ligands also have good selectivity, the alkaloid brucine and derivatives are selective for M(1), M(3) and M(4) receptors. Muscarinic toxins have been used in several types of experiments. For instance radioactively labeled M(1) and M(4) selective toxins were used in autoradiography of hippocampus from Alzheimer patients. One significant change in the receptor content was detected in one region of the hippocampus, dentate gyrus, where M(4) receptors were reduced by 50% in patients as compared to age-matched controls. Hippocampus is essential for memory consolidation. M(4) receptors in dentate gyrus may play a role, since they decreased in Alzheimers disease which destroys the memory. Another indication of the role of M(4) receptors for memory is that injection of the M(4) selective antagonist muscarinic toxin 3 (M(4)-toxin 1) into rat hippocampus produced amnesia.

Amino acid sequence of a snake venom toxin that binds to the muscarinic acetylcholine receptor

The green mamba, Dendroaspis angusticeps, has two protein toxins that bind to the muscarinic acetylcholine receptor. The sequence of muscarinic toxin 2 was determined with an automatic gas phase sequencer. The C-terminal residue is Asp as determined by hydrazinolysis and amino acid analysis. Toxin 2 has 65 amino acid residues and a formula weight of 7040. It is homologous to a large number of other snake venom toxins as short alpha-neurotoxins, cardiotoxins/cytotoxins and angusticeps-type toxins of mamba venoms. The sequence is confirmed in the accompanying article (Ducancel, F., Rowan, E.G., Cassart, E., Harvey, A. L., Menez, A. and Boulain, J.-C. Toxicon 29, 516-520, 1991).

Localization of M1 muscarinic receptors in rat brain using selective muscarinic toxin-1.

Mambas, African snakes of the genus Dendroaspis, produce several types of toxins that are of pharmacological interest. The novel muscarinic toxin-1 (MT-1), from the green mamba Dendroaspis angusticeps, binds specifically to muscarinic M1 receptors in homogenates of rat cerebral cortex. Iodination of the toxin, 125I-muscarinic toxin-1 (125I-MT-1), renders the toxin selective for M1 muscarinic receptors. Quantitative measurement of 125I-MT-1 autoradiography in rat brain sections indicated highest labeling in the nucleus accumbens, striatum, and dentate gyrus. High densities of 125I-MT-1 binding sites were located in the CA1 region of the hippocampus, frontal, and parietal cortices. Moderate densities of binding sites were seen in temporal cortex, and hippocampal subregions CA2, CA3, and CA4, whereas low labeling was observed in the cerebellum and spinal cord.

A snake toxin against muscarinic acetylcholine receptors: amino acid sequence, subtype specificity and effect on guinea-pig ileum.

The sequence of muscarinic toxin 1 (MT1) from Dendroaspis angusticeps (green mamba) was determined (66 amino acids, M(r) 7509). The central part, peptide 25-40, is rich in hydrophobic amino acids, which is a characteristic of muscarinic toxins. MT1 started to inhibit [3H]-NMS (N-methylscopolamine) binding to synaptosomal membranes of porcine brain (contains all five receptor subtypes) at about 1 nM and to membranes from pig heart muscle (only subtype m2) at about 1 microM. Binding of [3H]-AF-DX 384 to heart was inhibited with an IC50 of 14 microM and to brain in two steps. In the first step (IC50 = 32 nM) binding decreased by 37%, indicating that the toxin acted on m1 or m4 receptors, each accounting for about 40% of total receptor content. The second step was similar to the effect on heart. Pirenzepine inhibited binding of [125I]-MT1 to brain receptors with an IC50 of 6.5 nM, corresponding to a Ki of about 6 nM. Literature values of Ki for pirenzepine are 16-18 nM for m1 and > or = 120 mM for other subtypes. This indicates binding to m1 receptors. mM for other subtypes. This indicates binding to m1 receptors. [125I]-MT1 bound to brain with a Kd of 20 nM and a Hill coefficient of 1.0, i.e. one toxin molecule per receptor. In guinea-pig ileum, MT1 (670 nM) produced a rapid contraction, reversible by atropine. The toxin may be an agonist, but might also cause contraction by inducing acetylcholine release by a different mechanism.

Purification and sequence determination of a new muscarinic toxin (MT4) from the venom of the green mamba (Dendroaspis angusticeps).

A toxin which partially inhibited [3H]N-methylscopolamine binding to rat brain muscarinic receptors was purified from the venom of green mamba, Dendroaspis angusticeps. The N-terminal sequence (up to 45 amino acids) was determined by automated Edman degradation of the whole molecule. The complete sequence was elucidated after enzymatic cleavage with endoproteinase Arg-C or endoproteinase Lys-C and peptide fragments purification. The identity of the C-terminal amino acid was confirmed by hydrazinolysis. The new toxin (MT4) had eight half-cystines and 66 amino acids. It differed from muscarinic toxin MT1 by a single substitution in position 57 (arginine in MT1, histidine in MT4), proximal to the sixth half-cystine.

Two-step binding of green mamba toxin to muscarinic acetylcholine receptor

The mechanism of binding of toxin MT2 from venom of green mamba Dendroaspis angusticeps to muscarinic acetylcholine receptors from rat cerebral cortex was investigated by studying the kinetics of the toxin—receptor interaction. The muscarinic antagonist N‐methyl‐[3H]scopolamine was used as a ‘reporter’ ligand. Evidence for a mechanism of toxin—receptor interaction comprising at least two steps was obtained. Such a mechanism increases the potency of the toxin. The first step was fast with no competition between the toxin and the antagonist. The second step was slow with formation of a more stable toxin—receptor complex and inhibition of the antagonist binding. It is proposed that the snake toxin is a muscarinic agonist of slow action.

Kinetic evidence for different mechanisms of interaction of black mamba toxins MTα and MTβ with muscarinic receptors

Abstract By studying the influence of two toxins from the black mamba Dendroaspis polylepis on the kinetics of [ 3 H]- N -methylscopolamine binding to muscarinic acetylcholine receptors from rat cerebral cortex, it was revealed that these toxins, MTα and MTβ, interact with the receptors via kinetically distinct mechanisms. MTβ bound to receptors in a one-step, readily reversible process with the dissociation constant K d =5.3 μM. The binding mechanism of MTα was more complex, involving at least two consecutive steps. A fast receptor–toxin complex formation ( K T =3.8 μM) was followed by a slow process of isomerisation of this complex ( k i =1.8×10 −2 s −1 , half-time 39 s). A similar two-step interaction mechanism has been established for a related toxin, MT2 from the green mamba D. angusticeps ( K T =1.4 μM, k i =8.3×10 −4 s −1 , half-time 840 s). The slow isomerisation process delays the effect of MTα and MT2, but increases their apparent potency compared to toxins unable to induce the isomerisation process.