"Weak Toxin" from Naja kaouthia Is a Nontoxic Antagonist of alpha 7 and Muscle-type Nicotinic Acetylcholine Receptors*

Yuri N. UtkinDagger , Viktoriya V. KukhtinaDagger , Elena V. KryukovaDagger , Florence Chiodini§, Daniel Bertrand§, Christoph Methfessel, and Victor I. TsetlinDagger ||

From the Dagger  Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Str., V-437 Moscow GSP-7, 117997 Russia, the § Department of Physiology, Centre Medical Universitaire, 1 Rue Michel Servet, 1211 Geneva 4, Switzerland, and  Central Research Biophysics, Bayer AG, D-51368 Leverkusen, Germany

Received for publication, January 29, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A novel "weak toxin" (WTX) from Naja kaouthia snake venom competes with [125I]alpha -bungarotoxin for binding to the membrane-bound Torpedo californica acetylcholine receptor (AChR), with an IC50 of ~2.2 µM. In this respect, it is ~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 [125I]alpha -bungarotoxin from the Escherichia coli-expressed fusion protein containing the rat alpha 7 AChR N-terminal domain 1-208 preceded by glutathione S-transferase with IC50 values of 4.3 and 9.1 µM, respectively, whereas for neurotoxin II the IC50 value was >100 µM. Micromolar concentrations of WTX inhibited acetylcholine-activated currents in Xenopus oocyte-expressed rat muscle AChR and human and rat alpha 7 AChRs, inhibiting the latter most efficiently (IC50 of ~8.3 µM). 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 alpha 7 AChRs, and is closer to the long alpha -neurotoxins, which have comparable potency against the above-mentioned AChR types.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The first representative of "weak toxins," characterized by low toxicity, was isolated from Naja melanoleuca snake venom and sequenced in 1975 (1). Toxins of this type were later referred to as melanoleuca (2) or miscellaneous-type (3) toxins. In protein data banks (e.g. Swiss-Prot) they are classified together with some other toxins in the group of weak toxins. At present, around 15 amino acid sequences of such toxins are known. They consist of 62-68 amino acid residues, contain five disulfide bridges, and belong to the group of snake venom "three-fingered" toxins, whose characteristic feature is the presence of three disulfide-confined loops ("fingers") (4, 5). However, most other three-fingered toxins, including cardiotoxins, muscarinic toxins, acetylcholinesterase inhibitors, and the so-called short chain alpha -neurotoxins, contain four disulfide bridges. A fifth disulfide bridge is present in the long chain alpha -neurotoxins, such as alpha -bungarotoxin (alpha Bgt)1 from Bungarus multicinctus or alpha -cobratoxin from Naja kaouthia (CTX) and kappa -bungarotoxin (kappa Bgt), which together with short alpha -neurotoxins are potent antagonists of different classes of nicotinic acetylcholine receptors (AChRs) (for reviews see Refs. 6-8). In the long chain alpha - and kappa -neurotoxins the fifth disulfide bridge is located in the central loop II, whereas in weak toxins the additional disulfide is in the N-terminal loop I (see Fig. 1B). The available data show that residues of loops I, II, and III of alpha -neurotoxins participate in binding to AChRs (for reviews see Refs. 4, 5, and 7). However, depending on the type of alpha -neurotoxin (short or long) and AChR (muscle-type or neuronal alpha 7), the role of particular loops may vary. For example, there are numerous data demonstrating the involvement of identified residues in the central loop II of short chain and long chain neurotoxins in binding both to muscle-type (9-12) and alpha 7 AChRs (13). Interestingly, a fifth disulfide in loop II of the long alpha -neurotoxins is essential for binding to alpha 7 AChR but not to the Torpedo AChR (14). The residues of loop I in short alpha -neurotoxins were shown to be involved in binding to the Torpedo receptor (15), whereas loop I in long alpha -neurotoxins appears to be less important for the interaction with Torpedo and alpha 7 AChRs (11, 13).

In the present work, we decided to check whether a weak toxin isolated from N. kaouthia venom (designated WTX) acts on AChRs. WTX was found to bind to the membrane-bound Torpedo californica AChR, although much less efficiently than a short chain neurotoxin, NT-II, or a long one, CTX. However, WTX and CTX were virtually equipotent in binding to the soluble N-terminal domain of the rat alpha 7 AChR expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). Subsequent electrophysiological experiments revealed that in the micromolar range WTX blocked human and rat alpha 7 AChRs as well as rat muscle AChR (16).

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

WTX and Other Toxins

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).

CTX was obtained from N. kaouthia venom as described previously (18). NT-II was purified from the N. oxiana venom by gel filtration (under the same conditions as those used for N. kaouthia venom) followed by ion exchange chromatography on Bio Rex 70 in a gradient of ammonium acetate from 0.05 to 0.3 M (pH 7.5). alpha Bgt was purchased from Sigma. Radioactive [125I]alpha Bgt was prepared and purified as described previously (19).

AChR Preparations

Membrane-bound nicotinic acetylcholine receptor from T. californica electric organ was kindly provided by Prof. F. Hucho. A soluble fusion protein consisting of the N-terminal domain 1-208 of rat alpha 7 AChR preceded by GST, whose denatured form was first mentioned in Ref. 20, was isolated after expression of the respective cDNA in E. coli (data to be published elsewhere).2 Briefly, a gene fragment corresponding to the amino acids 1-208 was cloned into the pGEX-KG vector (21) at the BamHI/HinIII sites, and the recombinant plasmids were used to transform the E. coli strain JM107. The inclusion bodies containing the target protein were washed extensively with 1% Triton X-100 in the presence of 0.5 M NaCl, then dissolved in 8 M urea, 1 mM dithiothreitol, subsequently refolded at 10 °C in 20 mM Tris-HCl, pH 9.5, 0.1% CHAPS, concentrated by ultrafiltration, and then dialyzed against 10 mM Tris-HCl, pH 8.0, 0.1% CHAPS.

Binding Experiments

Membrane-bound AChR from T. californica-- Varying concentrations of competitors were added to 50 µl of membrane suspension (25 µg of protein/ml, 50 mM Tris-HCl buffer, pH 8.0), and the mixture was incubated for 1 h at room temperature in a total volume of 195 µl. Then 5 µl of 0.4 µM [125I]alpha Bgt (specific activity 45 Ci/mmol) were added, and the samples were incubated for 1 h more. The mixture was quickly filtered through Whatman GF/F filters (preincubated in 0.25% polyethylenimine for at least 2 h), the filters were washed four times with 1 ml of 50 mM Tris-HCl buffer, pH 8.0, and the radioactivity was determined in an Ultragamma gamma -counter (Amersham Pharmacia Biotech).

Fusion Protein-- A solution of GST-alpha 7-(1-208) protein (17 µg/ml, pH 8.0, 0.1% CHAPS) was incubated with different concentrations of competitors in a final volume of 190 µl for 1 h at room temperature. Then 10 µl of 0.4 µM [125I]alpha Bgt were added, and the mixture was incubated for 1 h more. The unbound [125I]alpha Bgt was removed by fast filtration through DE-81 filters (Whatman), and the filters were washed four times with 1 ml of 50 mM Tris-HCl buffer, pH 8.0, containing 0.1% Triton X-100 and counted.

Immunological Assays

CTX was reduced under denaturing conditions, carboxymethylated, and used to prepare a keyhole limpet hemocyanin conjugate, which was employed to immunize rabbits, and to obtain polyclonal antibodies by a standard procedure (22). An immunoglobulin fraction, obtained from the antiserum by fractional precipitation with ammonium sulfate, was dissolved in phosphate-buffered saline, dialyzed against this buffer, lyophilized, and stored at -80 °C. Enzyme-linked immunosorbent assay was performed in 96-well Linbro E.I.A. microtitration plates (Flow Laboratories) as in Ref. 23, using immunoglobulins of nonimmunized rabbits as a control. It was found that antibodies reacted with comparable efficiency with the reduced, carboxymethylated CTX and native CTX; the titers were 1:64,000 and 1:50,000, respectively.

Reconstitution in Xenopus Oocytes

Xenopus oocytes were isolated and prepared as previously described (24). A volume of 10 nl containing 2 ng of expression vector cDNA was injected into the nucleus the day following the oocyte isolation. To allow good protein expression levels, oocytes were kept for 2-3 days prior to recording. Each oocyte was placed in a separate well of a 96-well microtiter plate (Nunc) at 18 °C. Oocytes were incubated in Barth's solution, a standard medium for maintaining Xenopus oocytes in vitro, which consists of the following (in mM): NaCl 88, KCl 1, NaHCO3 2.4, HEPES 10, MgSO4 0.82, Ca(NO3)2 0.33, CaCl2 0.41. pH was adjusted to 7.4 with NaOH. To minimize contamination, the medium was filtered at 0.2 µm and supplemented with antibiotics (20 µg/ml kanamycin, 100 units/ml penicillin, and 100 µg/ml streptomycin or 50 µg/ml gentamicin).

Electrophysiology Experiments

Electrophysiological recordings were made using a dual electrode voltage clamp (TEC 00, NPI Electronic GmbH, Tamm, Germany) as described previously (25, 26). Cells were placed in a plexiglass chamber, voltage-clamped at -80 mV, and superfused with normal frog Ringer's solution (in mM: NaCl 115, KCl 2.5, CaCl2 1.8, HEPES 10, pH 7.2, adjusted with NaOH). ACh stock solution (0.1 M) was kept frozen and added to the test solutions on the day of the experiment. Cells were treated with WTX by incubating in the measuring chamber with a WTX-containing normal frog Ringer's solution for 20 min with perfusion stopped.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The WTX isolated by sequential gel filtration, ion exchange, and reverse-phase chromatography is homogeneous by the criteria of the latter and has a molecular mass of 7613 Da (Fig. 1A). However, this mass is larger than that (7483 Da) calculated from the published sequence of the weak toxin CM-9a from N. kaouthia venom (2). There are no tryptophan residues in CM-9a, whereas determination of the primary structure of WTX (17) detected a Trp residue in position 36 and also revealed two other differences from the CM-9a sequence: Lys-50 instead of Tyr and Tyr-52 instead of Lys (17). The molecular mass calculated for this sequence practically coincides with that found experimentally. Therefore, WTX, whose sequence is depicted in Fig. 1B, can be considered as a new homologue of CM-9a. When tested for a capacity to compete with [125I]alpha Bgt for binding to the membrane-bound T. californica AChR, WTX was found to be about 300 times less potent than CTX and NT-II, the long chain and short chain alpha -neurotoxins (Fig. 2A and Table I).


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Fig. 1.   Analytical characteristics and primary structure of weak toxin WTX. A, reverse-phase HPLC of WTX on a Vydac C18 column (4.6 × 250 mm) in an acetonitrile concentration gradient (from 15 to 45% in 30 min) in water in the presence of 0.1% trifluoroacetic acid. The flow rate was 1 ml/min. Inset, matrix-assisted laser desorption ionization mass spectrum of WTX. B, amino acid sequence of WTX. Shown against a black background is Trp-36, absent in weak toxin CM-9a. The residue numbers are given for the fragment 50-52, wherein the Lys-50 and Tyr-52 are interchanged in CM-9a, as well as for Cys-6 and Cys-11, characteristic for weak toxins. The polypeptide backbone of WTX is depicted in a typical three-fingered pattern, with the respective loops (fingers) marked I-III.


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Fig. 2.   Inhibition of [125I]alpha Bgt binding to the AChR-enriched membrane preparations of the T. californica electric organ (A) and to the solubilized fusion protein GST-alpha -(1-208) (B) by the snake venom toxins WTX (), CTX (black-square), and NT-II (black-triangle) and by alpha -conotoxin ImI (black-down-triangle ). Symbols black-diamond  and * refer to the experiments done using WTX subjected to one run and three runs of ion exchange HPLC (see Fig. 4), respectively. [C], toxin concentration (M).

                              
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Table I
Binding of polypeptide and peptide toxins to the membrane-bound Torpedo AChR and GST-alpha 7-(1-208) fusion protein

In addition to the muscle-type AChRs, the long neurotoxins alpha Bgt and CTX also bind to neuronal alpha 7 AChR. To assess this possibility for WTX as well, we used a bacterially expressed protein containing the N-terminal domain (amino acid residues 1-208) of the rat brain alpha 7 receptor preceded by GST. This chimera faithfully reproduces a number of alpha 7 AChR features (Fig. 2B and Table I). It interacts with alpha Bgt and CTX as well as with the alpha 7-targeting alpha -conotoxin ImI but practically does not bind either NT-II, a short-chain alpha -neurotoxin, or alpha -conotoxin G1 acting on the Torpedo and other muscle-type AChRs. In competition with [125I]alpha Bgt for binding to the chimerical protein, WTX was as active as CTX (Table I).

To test whether WTX binding can also lead to a functional block of the native alpha 7 AChRs, electrophysiological investigations on the alpha 7 AChR expressed in Xenopus oocytes were performed. Incubation for 20 min in the presence of 10 µM WTX caused an almost complete block of the subsequent ACh-evoked current (Fig. 3A). Such an inhibitory potency is much weaker than that of CTX or alpha Bgt, which block alpha 7 AChRs in the nanomolar range (13, 27, 28), but is of the same order of magnitude as the IC50 of alpha -conotoxin ImI in blocking the rat alpha 7 AChR (26, 29). We decided to check whether WTX is closer to alpha Bgt/CTX or to alpha -conotoxin ImI in the reversibility of its blocking effect on the rat alpha 7 AChR.


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Fig. 3.   Inhibition of AChRs expressed in Xenopus oocytes by WTX and CTX from N. kaouthia snake venom. The currents were evoked by short applications of ACh (50 µM, 4 s) to oocytes before (control) and after 20 min of incubation with toxins. Cells were held throughout the experiments at -80 mV. In A and B, the slow recovery of the response following washout is also shown. A, rat alpha 7 AChR with the initial sample of WTX before further purification; B, rat alpha 7 AChR with a repurified sample of WTX; C and D, human alpha 7 AChR and rat muscle AChR, respectively, both using repurified WTX; E, inhibition of the human alpha 7 AChR by 10 nM CTX.

Superfusion with control medium following WTX blockade showed very slow recovery, taking more than 1 h (Fig. 3A). It is similar to the slow dissociation of alpha Bgt (29) or CTX (see Ref. 30 and Fig. 6), whereas alpha -conotoxin ImI could be washed out almost completely in 10 min.

In view of the facts that WTX is washed out from oocytes as slowly as CTX but in terms of acting concentrations is about 1000-fold less potent, it was necessary to address the possibility that the effects on alpha 7 AChRs in oocytes ascribed to WTX in fact might have arisen if traces of CTX, even as low as 0.1%, were present in the WTX preparations. Therefore, additional experiments were done to check and, if necessary, to increase the degree of purity of WTX.

Fig. 1A shows only one peak in the mass spectrum of WTX purified by reverse-phase HPLC. No sequences other than that of WTX were detected on Edman degradation (17). However, matrix-assisted laser desorption ionization analysis of a model 1:1000 mixture of CTX:WTX failed to detect the peak of CTX. Fig. 4 shows chromatography of ~4 µg of CTX (trace 3), which would correspond to the 0.1% content in 4 mg of WTX. It is clear that such a small amount can be reliably detected under the chosen conditions. Fig. 4, trace 1 corresponds to purification of 4.2 mg of WTX from the batch used for structure determination and in the biological activity experiments described above. In addition to the huge peak of WTX itself, several minor peaks (each 0.1% or less) can be distinguished, including one coinciding with the position of CTX.


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Fig. 4.   Chromatography of WTX (4.2 mg, solid line, trace 1) and CTX (3.9 µg, dotted line, trace 3) on a TSK SP-5PW column (70 × 7.5 mm) in an ammonium acetate concentration gradient (pH 6.5) from 5 mM to 1 M in 100 min. The flow rate was 0.7 ml/min. Dashed line, trace 2, rechromatography of the WTX fractions eluting between 45 and 65 min on trace 1.

When the pooled fractions of the repurified WTX (eluting between 45 and 65 min) were lyophilized and applied again to the same column (Fig. 4, trace 2), no peak was detected at the position of CTX. Some other minor contaminating peaks were still present, which was not surprising in view of the relatively large amount of WTX applied onto the column and the extremely high sensitivity of UV detection.

We checked for the presence of CTX traces in the repurified WTX with the aid of polyclonal antibodies raised against reduced and carboxymethylated CTX. As seen from Fig. 5, these antibodies allow the detection of very low concentrations of CTX. Although at high concentrations (>0.01 mg/ml) WTX also reacts with these antibodies, the difference in the affinities makes possible the estimation of the upper limit of putative CTX admixture in the repurified WTX. A virtually complete inhibition of the alpha 7 AChR expressed in oocytes was observed at 20 µM (0.15 mg/ml) repurified WTX (see below, Figs. 3B and 7). As seen from Fig. 5, to this concentration of WTX corresponds an OD of ~0.16. If we assume that it is only the CTX admixture in WTX that interacts with antibodies, this amount should be as low as ~8.8 × 10-6 mg/ml (See Fig. 5), i.e. not more than 0.006% of the WTX. Moreover, if there is a slight cross-reactivity of antibodies with WTX, the admixture should be even lower, if present at all.


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Fig. 5.   Enzyme-linked immunosorbent assay titration of CTX () and WTX (black-square). Antibody concentration was 20 nM. [C], toxin concentration (mg/ml). Each point represents data of six independent determinations in two repeats.

Thus, immunological data confirmed that after additional purification by ion exchange HPLC, there are no measurable traces of CTX in WTX, at least in the range that would force one to ascribe the biological activity of WTX to CTX admixtures. Some of the binding experiments (Fig. 2) and more detailed studies in oocytes (Figs. 3, 6, and 7) were done with the repurified samples of WTX.


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Fig. 6.   Progress of recovery of current responses for several neurotoxins. After 10-20 min of incubation with toxin, cells were again continually superfused with Frog Ringer and challenged with 50 µM ACh for 4-s applications at 5-min intervals to monitor the recovery of the ACh-induced current response. Vertical bars show current signal amplitudes normalized to the amplitude of the initial response of the cell to 50 µM ACh. Black squares indicate 50 µM ACh applications at 5-min intervals. The two upper diagrams correspond to the WTX preparations before and after additional purification (Fig. 4), respectively.


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Fig. 7.   Concentration dependences for the WTX inhibition of the ACh-activated currents in rat (black-square) and human () alpha 7 AChRs expressed in Xenopus oocytes.

Additional purification had no effect on the WTX binding either to the Torpedo membranes or to alpha 7 AChR fusion protein. In Fig. 2 the experimental points for the repurified WTX (marked as diamonds and asterisks) fit the displacement curves obtained with the preparation that did not undergo such a treatment.

Electrophysiological experiments on the additionally purified WTX gave similar results as those described above; the WTX concentration required to achieve a virtually complete block was 20 µM (cf. Fig. 3, A and B). It is also seen (Fig. 3C) that at 20 µM WTX reduces by about 60% the ACh-induced current in the human alpha 7 AChR. In the same concentration range it efficiently blocks the rat muscle AChR expressed in Xenopus oocytes (Fig. 3D). As mentioned above, long chain alpha -neurotoxins like alpha Bgt or CTX block the alpha 7 and muscle AChR in the nanomolar range, as illustrated in Fig. 3E for CTX action on the human alpha 7 AChR.

From the washout experiments shown in Fig. 3, A and B, and, in more detail, in Fig. 6, it is clear that additional purification did not affect the virtual irreversibility of the WTX action. In this respect, it is practically indistinguishable from alpha Bgt or CTX and differs greatly from alpha -conotoxin ImI, which can be washed out in minutes (Fig. 6).

Fig. 7 shows the dose-response data illustrating the WTX action on the rat and human alpha 7 AChRs. Accurate quantitative measurements of these blocking effects on oocytes are difficult, especially at low toxin concentrations, because of the long incubation times required for the WTX to manifest its activity. We were able to estimate the IC50 value for the WTX action on the rat alpha 7 AChR as 8.3 µM, whereas for the qualitatively less potent action on human alpha 7 AChR we can give a very rough estimation of the IC50 value as ~15 µM.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our initial purpose was to isolate from the N. kaouthia snake venom a weak toxin, CM-9a, the only weak toxin previously found in this venom (2), and try to find its biological target. However, because the determined molecular mass of the corresponding fraction, designated by us as WTX, differed by 130 Da from that calculated from the published amino acid sequence of CM-9a (2), we had to determine the complete primary structure of WTX (17). Whereas CM-9a, like all previously described weak toxins from cobra venoms, contains no tryptophans, WTX has a Trp residue at position 36 and differs from CM-9a in two other positions: Lys-50/Tyr-50 and Tyr-52/Lys-52 (see Fig. 1). Therefore, WTX appears to be a new homologue of CM-9a. These minor alterations in the primary structure may simply be due to collecting the venoms from different snake populations. This structural difference can, however, influence the biological activity of the toxins. It is known that the toxicity (LD50) of weak toxins can vary from about 5 to 80 mg/kg (2, 31). We did not determine the LD50 for WTX, but in doses up to 2 mg/kg (intravenous injection) it was nontoxic to rats. Interestingly, a recently isolated gamma -bungarotoxin, structurally related to weak toxins (32), has an LD50 of 0.15 mg/kg, which is comparable with those of alpha -neurotoxins, but its molecular target has not been yet characterized.

As an attempt to define the biological targets for weak toxins, we examined the activity of WTX against two types of nicotinic acetylcholine receptors. As seen from Fig. 2A and Table I, WTX binds specifically to the membrane-bound Torpedo AChR, but about 300 times less effectively than CTX or NT-II. A comparison of WTX with these two alpha -neurotoxins was done also with an alpha 7 AChR-related system using a fusion protein, GST-alpha 7-(1-208). Although this protein binds CTX with only a moderate affinity (Fig. 2B and Table I), the following data allow us to consider it to be an appropriate model of the rat alpha 7 AChR ligand-binding domain. It does discriminate, similarly to the intact alpha 7 AChR, between the long and short alpha -neurotoxins (no inhibition of [125I]alpha Bgt binding by short neurotoxin NT-II at 100 µM) as well as between the muscle-type and neuronal alpha -conotoxins (no inhibition by alpha -conotoxin G1 at 100 µM and an IC50 of 42.6 µM for neuronal alpha -conotoxin ImI). Under these circumstances, the comparable inhibitory potencies of WTX and CTX (IC50 of 4.3 and 9.1 µM, respectively) allowed us to anticipate that WTX would bind specifically to the native alpha 7 AChRs.

Indeed, subsequent electrophysiological recordings demonstrated the capability of WTX to inhibit functional responses of alpha 7 AChRs expressed in Xenopus oocytes (Figs. 3, 6, and 7). Although WTX and CTX are virtually indistinguishable in their binding to the fusion protein GST-alpha 7-(1-208) (Fig. 2B), WTX is about 1000-fold less potent than CTX in blocking the ACh current on the native alpha 7 receptors heterologously expressed in Xenopus oocytes. As described under "Results," additional purification (Fig. 4) and immunological control experiments (Fig. 5) allowed us to rule out the possibility that the observed WTX effects on the ACh currents in oocytes might be caused by traces of CTX, if it were present even in as small amounts as 0.1%.

During the revision of this paper, a weak toxin from the Naja naja atra snake venom was shown to block cholinergic transmission in frog muscle preparations in the micromolar range (33), and a similar activity on a chick muscle was reported for a weak toxin from Bungarus candidus (34). However, these two weak toxins were not tested against alpha 7 AChRs, and the authors did not set a task to rule out the presence of trace amounts of the respective alpha -neurotoxins.

WTX, despite acting on the alpha 7 AChR at micromolar rather than nanomolar concentrations, as do alpha Bgt or CTX, is very similar to these two long chain alpha -neurotoxins in the persistency of the produced block (Fig. 6). In this respect it differs from alpha -conotoxin ImI and its analogs, which are also active in the micromolar range but can be washed out within minutes (26, 29) (see Fig. 6).

A number of amino acid residues and certain conformational motifs essential for binding to respective AChRs have been identified both in alpha -neurotoxins and alpha -conotoxins (4, 5, 11, 13, 35). The additional disulfide bridge in the CTX and in kappa -bungarotoxin is a prerequisite for their binding to neuronal AChRs, that is to alpha 7 and alpha 3beta 2, respectively (14, 36). This disulfide fixes a helix-like scaffold in the alpha -neurotoxin central loop II (37). A similar scaffold could be distinguished in alpha -conotoxin ImI acting on the alpha 7 AChR as well as in the other alpha -conotoxins targeting the neuronal AChRs, but not in alpha -conotoxin G1 blocking the Torpedo and muscle AChRs (38). Interestingly, there are no alpha -helices or helix-like segments in the N-terminal loop I of the three-fingered toxins. A helix was found in the CD59 protein (39), which, like other members of the Ly-6 family of immune system proteins (40, 41), has a fifth disulfide in the N-terminal loop I, rather than in the loop II as do the long-type alpha -neurotoxins. The "endogenous toxin" lynx-1 recently found in the nervous system (42) was supposed to have a helix turn in loop I, but the conclusion was based only on modeling studies. However, although confirming by x-ray analysis the beta -structure-dominated three-fingered motif in bucandin, a presynaptic neurotoxin, no helical turns were found in its loop I, containing the additional disulfide (43). Only beta -structure was detected by 1H-NMR studies of WTX (44). It seems that specificity and selectivity of a particular three-fingered toxin toward various AChRs or other targets depends not only on the chemical nature and distribution of functional groups (charged, hydrophobic, etc.) along the three-fingered scaffold, but also on the differences in the conformation and conformational mobility of those loops. In particular, long times required to restore the sensitivity of the alpha 7 AChR to Ach after applying and washing out WTX might be associated with the conformational heterogeneity of WTX (44) and conformational changes that WTX might undergo itself on binding to the receptor or induce in the latter. Noteworthy allosteric effects of antagonists and agonists, some of them influencing the channel moiety, were described for the Torpedo AChR (45, 46).

In summary, the results obtained suggest that the biological target of WTX, a member of the family of weak toxins, may be nicotinic AChRs. Interacting with the Torpedo, muscle, and alpha 7 AChRs at similar concentrations, WTX resembles the long chain alpha -neurotoxins more than the short ones in this respect. Although acting only at micromolar concentrations, WTX has an advantage of being practically nontoxic while producing a long lasting effect. In view of the pharmacological importance of neuronal AChRs (for review see Ref. 47), a search for selective and efficient markers and blockers of those receptors is still an important task. Our work shows that the three-fingered proteins of the "weak-toxin structural subtype" are promising in this respect.

    ACKNOWLEDGEMENTS

We are grateful to F. Hucho for helpful discussions and for providing the Torpedo membranes and to V. Witzemann, J. Patrick, and B. Weingärtner for kindly providing the cloned rat muscle AChR cDNA, rat alpha 7 cDNA, and human alpha 7 cDNA, respectively. We are also indebted to V. Starkov for breeding snakes and supplying their venoms and to N. Dergousova and E. Shibanova for preparing the alpha 7 fusion protein. We thank S. Bertrand for help with electrophysiological experiments. We are grateful to an anonymous referee, whose remarks stimulated additional experiments on WTX.

    FOOTNOTES

* This work was supported by the Russian Foundation for Basic Research (Grants 99-04-48798 and 00-04-48889) and by the Swiss National Science Foundation and the "Office Federal de l'Education et des Sciences." A preliminary account was presented at the XIIIth World Congress of the International Society on Toxinology, September 18-22, 2000, Paris, France.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. Tel. & Fax: 7 095 335 57 33; E-mail: vits@ibch.ru.

Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M100788200

2 N. I. Dergousova, E. D. Shibanova, I. E. Kasheverov, C. Methfessel, and V. I. Tsetlin, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: Bgt, bungarotoxin; CTX, alpha -cobratoxin; ACh, acetylcholine; AChR, nicotinic acetylcholine receptor; WTX, weak toxin from N. kaouthia; NT-II, neurotoxin II from Naja oxiana; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HPLC, high performance liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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