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

Yuri N Utkin,Viktoriya V Kukhtina,Elena V Kryukova,Florence Chiodini,Daniel Bertrand,Christoph Methfessel,Victor I Tsetlin

doi:10.1074/jbc.m100788200

Yuri N Utkin, Viktoriya V Kukhtina + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m100788200

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Abstract

A novel "weak toxin" (WTX) from Naja kaouthia snake venom competes with [(125)I]alpha-bungarotoxin for binding to the membrane-bound Torpedo californica acetylcholine receptor (AChR), with an IC(50) of approximately 2.2 microm. In this respect, it is approximately 300 times less potent than neurotoxin II from Naja oxiana and alpha-cobratoxin from N. kaouthia, representing short-type and long-type alpha-neurotoxins, respectively. WTX and alpha-cobratoxin displaced [(125)I]alpha-bungarotoxin from the Escherichia coli-expressed fusion protein containing the rat alpha7 AChR N-terminal domain 1-208 preceded by glutathione S-transferase with IC(50) values of 4.3 and 9.1 microm, respectively, whereas for neurotoxin II the IC(50) value was >100 microm. Micromolar concentrations of WTX inhibited acetylcholine-activated currents in Xenopus oocyte-expressed rat muscle AChR and human and rat alpha7 AChRs, inhibiting the latter most efficiently (IC(50) of approximately 8.3 microm). Thus, a virtually nontoxic "three-fingered" protein WTX, although differing from alpha-neurotoxins by an additional disulfide in the N-terminal loop, can be classified as a weak alpha-neurotoxin. It differs from the short chain alpha-neurotoxins, which potently block the muscle-type but not the alpha7 AChRs, and is closer to the long alpha-neurotoxins, which have comparable potency against the above-mentioned AChR types.

Highlights

A novel “weak toxin” (WTX) from Naja kaouthia snake venom competes with [125I]␣-bungarotoxin for binding to the membrane-bound Torpedo californica acetylcholine receptor (AChR), with an IC50 of ϳ2.2 ␮M
WTX and ␣-cobratoxin displaced [125I]␣bungarotoxin from the Escherichia coli-expressed fusion protein containing the rat ␣7 AChR N-terminal domain 1–208 preceded by glutathione S-transferase with IC50 values of 4.3 and 9.1 ␮M, respectively, whereas for neurotoxin II the IC50 value was >100 ␮M
Sulfide bridge is present in the long chain ␣-neurotoxins, such as ␣-bungarotoxin (␣Bgt)1 from Bungarus multicinctus or ␣-cobratoxin from Naja kaouthia (CTX) and ␬-bungarotoxin (␬Bgt), which together with short ␣-neurotoxins are potent antagonists of different classes of nicotinic acetylcholine receptors (AChRs)

Summary

EXPERIMENTAL PROCEDURES

WTX was purified from N. kaouthia as described previously [17]. The molecular mass of WTX was determined by matrix-assisted laser desorption ionization time of flight on a Vision 2000 (Thermo Bioanalysis Corp.) mass spectrometer. This preparation (See Fig. 1) was used for primary structure determination [17] and for preliminary testing of biological activity. Further experiments were done on the WTX preparations obtained after additional purification steps by ion exchange chromatography on a TSK SP-5PW column (see Fig. 4)

The abbreviations used are

RESULTS

DISCUSSION