Structure and Action of Polypeptide Axonal Toxins

Blumenthal, Kenneth

Abstract

The marine worm Cerebratulus lacteus contains a group of polypeptide neurotoxins which greatly prolong the repolarization phase of the action potential in crustacean neurons but are inactive upon squid giant axon, desheathed frog sciatic nerve, and garfish olfactory nerve. A toxin with similar species specificity is synthesized by the anemone Condylactis gigantis. The American scorpion Centuroides contains a number of toxins which lack such selectivity but, like the two former proteins, exert their effect via interaction with axonal sodium and/or potassium channels. The ionic and molecular mechanisms of action of these toxins will be investigated by a combination of electrophysiologic and biochemical techniques. We shall test our hypothesis that these polypeptides bind to specific membrane receptor protein(s) associated with the sodium or potassium channels. Electrophysiologic actions of the toxins will be investigated by standard microelectrode and voltage clamp techniques. The primary structure of Cerebratulus toxin B-II and the most abundant Condylactis toxin will be determined and the secondary structures of native and modified toxins probed by circular dichroism and Raman spectroscopy. Amino acid residues involved in toxin-receptor interactions will be determined by selective chemical modification and by controlled protease cleavage at the N- and C-termini of the proteins. The toxicity of the resulting toxin derivatives relative to the native toxins will be assessed by micro-electrode experiments as well as by quantal bioassay. We shall prepare highly radioactive yet pharmacologically active toxin derivatives suitable for estimating the receptor density, for determination of the equilibrium dissociation constant of the toxin-receptor complex, and for solubilization, purification, and characterization thereof.