“Weak Toxin” from Naja kaouthia Is a Nontoxic Antagonist of α7 and Muscle-type Nicotinic Acetylcholine Receptors

Abstract

A novel "weak toxin" (WTX) from Naja kaouthia snake venom competes with [125 I]␣-bungarotoxin for binding to the membrane-bound Torpedo californica acetylcholine receptor (AChR), with an IC 50 of ϳ2.2 M. In this respect, it is ϳ300 times less potent than neurotoxin II from Naja oxiana and ␣-cobratoxin from N. kaouthia, representing short-type and long-type ␣-neurotoxins, respectively. WTX and ␣-cobratoxin displaced [125 I]␣-bungarotoxin from the Escherichia coli-expressed fusion protein containing the rat ␣7 AChR N-terminal domain 1-208 preceded by glutathione S-transferase with IC 50 values of 4.3 and 9.1 M, respectively, whereas for neurotoxin II the IC 50 value was >100 M. Micromolar concentrations of WTX inhibited acetylcholine-activated currents in Xenopus oocyte-expressed rat muscle AChR and human and rat ␣7 AChRs, inhibiting the latter most efficiently (IC 50 of ϳ8.3 M). Thus, a virtually nontoxic "three-fingered" protein WTX, although differing from ␣-neurotoxins by an additional disulfide in the N-terminal loop, can be classified as a weak ␣-neurotoxin. It differs from the short chain ␣-neurotoxins, which potently block the muscle-type but not the ␣7 AChRs, and is closer to the long ␣-neurotoxins, which have comparable potency against the above-mentioned AChR types.

Water-soluble LYNX1 Residues Important for Interaction with Muscle-type and/or Neuronal Nicotinic Receptors

“…One such toxin, candoxin from Bungarus candidus, blocks both the muscle and ␣7 nAChRs with nanomolar affinity (35), whereas another non-conventional toxin, weak toxin from the cobra Naja kaouthia, acts on these receptors only with micromolar affinity (36). Thus, ws-LYNX1 in terms of affinities for nAChRs is close to weak toxin, but this toxin still shows virtual irreversibility of action (36). Interestingly, a common feature of ws-LYNX1 and weak toxin is their action on muscarinic acetylcholine receptors (3, 37).…”

Three-Finger α-Neurotoxins and the Nicotinic Acetylcholine Receptor, Forty Years On

“…However, electrophysiological studies have revealed that a -bungarotoxin (IC 50 approximately 5 nM) produced an irreversible block of muscle (a 1) 2 b1gd receptors (68), whereas candoxin (IC 50 approximately 10 nM) produced a reversible blockade of the same receptor (58). Furthermore, WTX (Naja kaouthia), a non-conventional toxin that is structurally similar to candoxin but a 1000-fold weaker antagonist of muscle nAChRs, is almost irreversible in its action (54). Therefore, the reversibility of a -neurotoxin action at the neuromuscular junction is not always a reflection of their binding affinity to the receptor and may perhaps be associated with a specific area of interaction on the toxin molecule, distinct from the receptor recognition site (67).…”

“…Short-chain a -neurotoxins as well as atypical longchain toxins (Lc-a and Lc-b from Laticauda colubrina), which lack this fifth disulfide bridge and non-conventional toxins WTX (Naja kaouthia) and Wntx-5 (Naja sputatrix), both of which have the fifth disulfide bridge in loop I and lack the loop II helical conformation, have weak affinity, in micromolar concentrations (K d approximately 3 -22 mM) at best, for the neuronal a 7 nAChR (50,54,56). Interestingly, candoxin (Bungarus candidus) is the first known toxin that lacks this critical helix-like conformation cyclized by the fifth disulfide bridge in loop II and yet blocks neuronal a 7 nAChRs in low nanomolar concentrations (58).…”

“…1). Some studies have suggested that non-conventional toxins are typically characterized by a lower order of toxicity (LD 50 varying from approximately 5 -80 mg / kg) (25,54) as opposed to prototype a -neurotoxins (LD 50 approximately 0.04 -0.3 mg / kg) (55), and because of this, they are also referred to as weak toxins (54). However, while some non-conventional toxins from cobra venoms (e.g., WTX (Naja kaouthia) (54) and Wntx-5 (Naja sputatrix) (56)) produced a weak inhibition of muscle (a1) 2 b1gd nAChRs in micromolar inhibitory concentrations, and candoxin from a krait venom (Bungarus candidus) was a potent inhibitor of muscle (a1) 2 b1gd nAChRs in low nanomolar (IC 50 approximately 10 nM) inhibitory concentrations (57).…”

Solution structure of candoxin, a novel three-finger toxin from the venom of Bungarus candidus

“…To date, only two proteins with this disulfide bridge scaffold have been structurally characterized. They are bucandin (pdb: 1F94 (X-ray) and 1IJC (NMR)), another toxin from Bungarus candidus venom [35][36] and Cd59 (pdb: 1CDQ), which is a cell surface protein 37 .…”