Workshop: the use of muscarinic toxins in the study of muscarinic receptors.

One of the most interesting recent developments in the pharmacology of muscarinic receptors has been the finding of small proteins in the venoms of mamba snakes that bind with high affinity and selectivity to different subtypes of muscarinic receptors. In the workshop on muscarinic toxins, the practicalities of isolating, characterising and using these toxins as tools in the study of muscarinic receptors were discussed.

Effect of fasciculin on hydrolysis of neutral and choline esters by butyrylcholinesterase, cobra venom and chicken acetylcholinesterases.

Acetylcholinesterases (AChEs) very sensitive to fasciculin inhibition (KiS in picomolar range) have a distinctive group of aromatic amino acids in the peripheral region (Y70, Y121, W279 in Torpedo AChE). Enzymes that lack these amino acids like butyrylcholinesterases (BChEs) or one or two of them like cobra venom, insect and chicken AChEs are 1000 to 1,000,000 times less sensitive. Fasciculin is a non-competitive inhibitor of the hydrolysis of choline and neutral esters by very sensitive AChEs. For the other group of enzymes, differences arise according to the type of substrate. Fasciculin still behaves as a non-competitive inhibitor with choline esters. In contrast, hydrolysis of phenylacetate was unaffected or slightly increased with BChEs and a partial competitive inhibition was observed with cobra venom and chicken enzymes.

Fasciculin: modification of carboxyl groups and discussion of structure-activity relationship.

Norleucine methylester was coupled to carboxylates of fasciculin 2, a snake toxin that inhibits acetylcholinesterase (AChE). This neutralized negative charges but had no effect on the activity, suggesting that carboxyls do not participate in binding to AChE. Earlier results are discussed. Modification of three aromatic amino acids in the peripheral site of AChE, the binding site for fasciculin, decreased the affinity 100 to one million times. Neutralizing the charge of cationic groups of fasciculin lowered the affinity only three to seven times. A change in either the toxin or enzyme part of a binding site should have about the same effect. Since this was not so, it suggests that cationic groups of fasciculin do not bind to aromatic rings in the peripheral site.

Characterization of a potassium channel toxin from the Caribbean Sea anemone Stichodactyla helianthus.

A peptide toxin, ShK, that blocks voltage-dependent potassium channels was isolated from the whole body extract of the Caribbean sea anemone Stichodactyla helianthus. It competes with dendrotoxin I and alpha-dendrotoxin for binding to synaptosomal membranes of rat brain, facilities acetylcholine release at an avian neuromuscular junction and suppresses K+ currents in rat dorsal root ganglion neurones in culture. Its amino acid sequence is R1SCIDTIPKS10RCTAFQCKHS20MKYRLSFCRK30TCGTC35. There is no homology with other K+ channel-blocking peptides, except for BgK from the sea anemone Bunodosoma granulifera. ShK and BgK appear to be in a different structural class from other toxins affecting K+ channels.

Role of arginine residues for the activity of fasciculin.

The West African green mamba, Dendroaspis angusticeps, has two toxins, fasciculins, that are non-competitive inhibitors of acetylcholinesterase. Arginine residues of fasciculin 2 were modified with 1,2-cyclohexanedione. Two of these residues, Arg24 and Arg37, reacted very slowly or not at all. Modification of Arg28 reduced the activity only by 13%. Arg11 and Arg27 are unique for fasciculins; a comparison of the sequences of 175 snake toxins homologous to fasciculins showed that no other toxin has arginine in the corresponding positions. Modification of the two unique arginines had a large effect and decreased the activity by 73% (Arg11) and 85% (Arg27). This was apparently not due to structural perturbations, since the modification did not change the circular dichroic spectra. The two arginine residues probably participate in the binding to acetylcholinesterase. They are located on the same side of the toxin molecule and the distance between their alpha-carbons is 2.7 nm. This may indicate binding to sites that are far apart and suggests that fasciculin covers a large area of the enzyme.

Study of structure-activity relationship of fasciculin by acetylation of amino groups.

Dendroaspis angusticeps (green mamba) has two toxins, fasciculins, that are non-competitive inhibitors of acetylcholinesterase. Amino groups of fasciculin 2 were acetylated with acetic anhydride. The monoacetyl derivatives of the epsilon-amino groups (Lys 25, 32, 51 and 58) retained between 28 and 43% of the initial activity and that of the alpha-amino group 72%. Acetylation of Lys 25 that has the most reactive amino group decreased the activity by 65% apparently without producing structural perturbations, since the circular dichroism spectrum was not affected. The three-dimensional structure shows a cationic cluster formed by Lys 32, 51, Arg 24 and 28. A comparison of 175 sequences of homologous toxins shows that Lys 32 is unique for fasciculin. Acetylation of lysine residues in the cluster had a large effect and reduced the activity by 72% (Lys 32) and 57% (Lys 51). This suggests an important role for the cationic cluster. Lys 25 together with Lys 32 and 51 were, therefore, assumed to be in the active site.

A potassium channel toxin from the secretion of the sea anemone Bunodosoma granulifera. Isolation, amino acid sequence and biological activity.

A peptide toxin affecting potassium channels was isolated from the sea anemone Bunodosoma granulifera. It facilitates acetylcholine release at avian neuromuscular junctions, competes with dendrotoxin I, a probe for voltage-dependent potassium channels, for binding to synaptosomal membranes of rat brain with a Ki of 0.7 nM and suppresses K+ currents in rat dorsal root ganglion neurones in culture. It represents a new structural type of potassium channel toxin with the sequence V1RCDWFKETA10CRHAKSLGNC20RTSQKYRANC30AKTLQCC37 (M(r) 4275, three disulfides).