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

Abstract

kappa-Flavotoxin (kappa-FTX), a snake neurotoxin that is a selective antagonist 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]. Like the related snake toxin kappa-bungarotoxin (kappa-BTX), kappa-FTX binds with high affinity to alpha 3 subtypes of neuronal AChRs, even though there are distinct sequence differences between the two toxins. To further characterize the sequence regions of the neuronal AChR alpha 3 subunit involved in formation of the binding site for this family of kappa-neurotoxins, we investigated kappa-FTX binding to overlapping synthetic peptides screening the alpha 3 subunit sequence. A sequence region forming a "prototope" for kappa-FTX was identified within residues alpha 3 (51-70), confirming the suggestions of previous studies on the binding of kappa-BTX to the alpha 3 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 region is involved in formation of a cholinergic site. Single residue substituted analogues, where each residue of the sequence alpha 3 (51-70) was sequentially replaced by a glycine, were used to identify the amino acid side chains involved in the interaction of this prototope with kappa-FTX.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-Function of Neuronal Nicotinic Acetylcholine Receptor Inhibitors Derived From Natural Toxins

“…However, a number of long-chain α-neurotoxins, including α-cbtx and α-bgtx, also inhibit neuronal α7 nAChRs with high affinity (Tsetlin and Hucho, 2004). Meanwhile, κ-neurotoxins preferably target neuronal α3-containing nAChRs (Chiappinelli, 1983;McLane et al, 1993).…”

Expression of cardiotoxin-2 gene Cloning, characterization and deletion analysis of the promoter

“…The primary sequence and the gene structure of these CTX genes were found to be similar to each other. The genes consist of three exons and two introns and resemble the gene structure of the a-neurotoxin, and k-neurotoxins [8,9]. Sequence analysis of the N. sputatrix CTX-3 gene promoter revealed the presence of two transcription initiation sites (TIS1 and TIS2), and two putative TATA box motifs and absence of a canonical upstream CCAAT element [6].…”

Neurotoxins Acting at Nicotinic Receptors

“…κ-bungarotoxin is also known as neuronal bungarotoxin owing to its high affinity for the neuronal subtypes of the nAChRs over muscle-type receptors, with highest affinity for the α3β2 subtype (Kd = 10 -8 M) and a Kd of 10 -5 M for the muscle-type receptors [62,63]. κ-bungarotoxin binds with lower affinity to muscle-type receptors (Kd = 10 -5 M) [62].…”

“…Reduction and dithiopyridylation of this disulfide lowers the affinity for the α7 nAChR (Kd = 12 × 10 -6 M), but does not affect the affinity for α3-containing subtypes (Kd = 0.35 × 10 -9 M) [56]. Another κ-neurotoxin, κ-flavitoxin, also binds with high affinity to α3 subtypes of neuronal AChRs [63]; moreover, nicotinic neurotransmission in autonomic ganglia is completely blocked by 50 nM κ-flavitoxin [41].…”