HOMOLOGOUS KAPPA-NEUROTOXINS EXHIBIT RESIDUE-SPECIFIC INTERACTIONS WITH THE ALPHA-3 SUBUNIT OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR - A COMPARISON OF THE STRUCTURAL REQUIREMENTS FOR KAPPA-BUNGAROTOXIN AND KAPPA-FLAVITOXIN BINDING

Kappa-flavotoxin (kappa-FTX), a snake neurotoxin that is a selective a ntagonist of certain neuronal nicotinic acetylcholine receptors (AChRs ), has recently been isolated and characterized [Grant, G. A., Frazier , M. W., & Chiappinelli, V. A. (1988) Biochemistry 27, 1532-1537). Lik e the related snake toxin kappa-bungarotoxin (kappa-BTX), kappa-FTX bi nds with high affinity to alpha3 subtypes of neuronal AChRs, even thou gh there are distinct sequence differences between the two toxins. To further characterize the sequence regions of the neuronal AChR alpha3 subunit involved in formation of the binding site for this family of k appa-neurotoxins, we investigated kappa-FTX binding to overlapping syn thetic peptides screening the alpha3 subunit sequence. A sequence regi on forming a ''prototope'' for kappa-FTX was identified within residue s alpha3(51-70), confirming the suggestions of previous studies on the binding of kappa-BTX to the alpha3 subunit [McLane, K. E., Tang, F., & Conti-Tronconi, B. M. (1990) J. Biol. Chem. 265,1537-1544] and alpha -bungarotoxin to the Torpedo AChR alpha subunit [Conti-Tronconi, B. M. , Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230] that this sequence re gion is involved in formation of a cholinergic site. Single residue su bstituted analogues, where each residue of the sequence alpha3(51-70) was sequentially replaced by a glycine, were used to identify the amin o acid side chains involved in the interaction of this prototope with kappa-FTX. Substitution of several aliphatic (L54, L56, and L65), arom atic (W55, W60, W67, and Y63), and positively charged (K57, K64, K66, and K68) residues drastically reduced the ability of the peptides to i nteract with kappa-FTX. Comparison of the results of the present study with those obtained previously for the binding to the same prototope of the related neuronal toxin, kappa-BTX, suggests that, like the inte raction of peripheral AChRs with the family of the alpha-neurotoxins, the interaction of neuronal AChRs with toxins of the kappa-neurotoxin family involves aromatic and aliphatic residues of general importance. Individual binding preference of specific toxins may involve charged amino acid residues at the toxin/AChR interface.