Snake {alpha}-Neurotoxin Binding Site on the Egyptian Cobra (Naja haje) Nicotinic Acetylcholine Receptor Is Conserved

Zoltan Takacs, Kirk C. Wilhelmsen and Steve Sorota

Department of Pharmacology, College of Physicians and Surgeons, Columbia University;
Department of Neurology and Ernest Gallo Clinic and Research Foundation, University of California at San Francisco


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Evolutionary success requires that animal venoms are targeted against phylogenetically conserved molecular structures of fundamental physiological processes. Species producing venoms must be resistant to their action. Venoms of Elapidae snakes (e.g., cobras, kraits) contain {alpha}-neurotoxins, represented by {alpha}-bungarotoxin ({alpha}-BTX) targeted against the nicotinic acetylcholine receptor (nAChR) of the neuromuscular junction. The model which presumes that cobras (Naja spp., Elapidae) have lost their binding site for conspecific {alpha}-neurotoxins because of the unique amino acid substitutions in their nAChR polypeptide backbone per se is incompatible with the evolutionary theory that (1) the molecular motifs forming the {alpha}-neurotoxin target site on the nAChR are fundamental for receptor structure and/or function, and (2) the {alpha}-neurotoxin target site is conserved among Chordata lineages. To test the hypothesis that the {alpha}-neurotoxin binding site is conserved in Elapidae snakes and to identify the mechanism of resistance against conspecific {alpha}-neurotoxins, we cloned the ligand binding domain of the Egyptian cobra (Naja haje) nAChR {alpha} subunit. When expressed as part of a functional Naja/mouse chimeric nAChR in Xenopus oocytes, this domain confers resistance against {alpha}-BTX but does not alter responses induced by the natural ligand acetylcholine. Further mutational analysis of the Naja/mouse nAChR demonstrated that an N-glycosylation signal in the ligand binding domain that is unique to N. haje is responsible for {alpha}-BTX resistance. However, when the N-glycosylation signal is eliminated, the nAChR containing the N. haje sequence is inhibited by {alpha}-BTX with a potency that is comparable to that in mammals. We conclude that the binding site for conspecific {alpha}-neurotoxin in Elapidae snakes is conserved in the nAChR ligand binding domain polypeptide backbone per se. This conclusion supports the hypothesis that animal toxins are targeted against evolutionarily conserved molecular motifs. Such conservation also calls for a revision of the present model of the {alpha}-BTX binding site. The approach described here can be used to identify the mechanism of resistance against conspecific venoms in other species and to characterize toxin-receptor coevolution.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
The biological function of snake venom neurotoxins is to immobilize potential prey and predator species. For evolutionary success, the molecular structure of the neurotoxin target site must be conserved in a phylogenetically wide spectrum of taxa among Chordata lineages. In addition, the same target site must be associated with fundamental physiological mechanisms, such as release or binding or degrading of the neurotransmitter at the neuromuscular junction, in order to provide a basis for the immediate and potentially lethal pharmacological effect of the neurotoxins.

Venoms of the Elapidae snakes (e.g., cobras, kraits) contain several postsynaptic polypeptide {alpha}-neurotoxins, such as {alpha}-bungarotoxin ({alpha}-BTX) isolated from the venom of the banded krait (Bungarus multicinctus) (Mebs et al. 1972Citation ). The common target of {alpha}-neurotoxins is the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel on the postsynaptic fold of the neuromuscular junction with the subunit stoichiometry of {alpha}2ß{gamma}{delta} (Karlin 1993Citation ). Upon binding to the nAChR, {alpha}-neurotoxins prevent the binding of the natural ligand acetylcholine (ACh) and the subsequent ACh-induced ion flow, resulting in a neuromuscular inhibition of the envenomated species. The primary binding site of {alpha}-BTX on Chordata nAChR has been localized within segment E172-F205 of the {alpha} subunits (Wilson, Lentz, and Hawrot 1985Citation ; Neumann et al. 1986a, 1986bCitation ; Barkas et al. 1987Citation ; Gotti et al. 1988Citation ; Wilson and Lentz 1988Citation ; Ohana and Gershoni 1990Citation ; Pearce and Hawrot 1990Citation ; Conti-Tronconi et al. 1991Citation ; McLane et al. 1991Citation ; Chaturvedi, Donnelly-Roberts, and Lentz 1992, 1993Citation ; McLane, Wu, and Conti-Tronconi 1994Citation ; Lentz 1995Citation ; Arias 2000Citation ). Specifically, {alpha} subunit residues H186, W187, V188, Y, T, or F at position 189, Y190, T191, C192, C193, P194, D195, P197, and D200 have been postulated to be the principal elements forming the {alpha}-BTX binding site in torpedo (Torpedo spp.), mouse (Mus musculus), and human (Mulac-Jericevic and Atassi 1986Citation ; Neumann et al. 1986aCitation ; Gotti et al. 1988Citation ; Mulac-Jericevic et al. 1988Citation ; Ohana and Gershoni 1990Citation ; Conti-Tronconi et al. 1991Citation ; McLane et al. 1991Citation ; Ohana et al. 1991Citation ; Barchan et al. 1992Citation ; Chaturvedi, Donnelly-Roberts, and Lentz 1992Citation ; Chaturvedi, Donnelly-Roberts, and Lentz 1993Citation ; Fuchs et al. 1993Citation ; McCormick et al. 1993Citation ; McLane, Wu, and Conti-Tronconi 1994Citation ; Kachalsky et al. 1995Citation ; Lentz 1995Citation ; Ackermann and Taylor 1997Citation ; Spura et al. 1999, 2000Citation ). In addition, a two-subsite model was proposed that requires aromatic amino acid residues at positions 187 and 189, plus the parallel presence of P194 and P197 in order to bind {alpha}-BTX (Barchan et al. 1995Citation ; Kachalsky et al. 1995Citation ).

The primary binding site of {alpha}-BTX, the {alpha} subunit segment E172-F205, is in the vicinity of {alpha}{gamma} and {alpha}{delta} subunit interfaces (Blount and Merlie 1989Citation ; Sine and Claudio 1991Citation ; Czajkowski and Karlin 1995Citation ). As predicted by the theory that the target sites of neurotoxins are associated with fundamental physiological mechanisms, this segment also includes major determinants for ACh binding, namely, residues Y190, C192, C193, and Y198 (Karlin 1993Citation ). All of these residues are conserved among Chordata lineages.

Venomous snakes exhibit a natural resistance to components of conspecific venoms. Considering the phylogenetic conservation and physiological relevance of the target site for venom components, the evolutionary and pharmacological basis for such resistance is rather interesting. To date, only one resistance strategy has been demonstrated, where components of pit viper (Crotalinae) venoms are neutralized by humoral factors present in conspecific blood plasma (Straight, Glenn, and Snyder 1976Citation ). Such a neutralization mechanism has not been established in Elapidae snakes (Ovadia and Kochva 1977Citation ), and serum-free neuromuscular preparations of snakes generally are insensitive to {alpha}-neurotoxins (Burden, Hartzell, and Yoshikami 1975Citation ; Liu, Xu, and Hsu 1990Citation ; Endo and Tamiya 1991Citation ). Earlier studies suggested that {alpha}-BTX resistance in cobras (Naja spp., Elapidae) was (1) due to the absence of the binding site because of the inherent amino acid substitutions in the polypeptide backbone of the nAChR {alpha} subunit per se (Neumann et al. 1989Citation ; McLane et al. 1991Citation ; Ohana et al. 1991Citation ; Barchan et al. 1992, 1995Citation ; Chaturvedi, Donnelly-Roberts, and Lentz 1992Citation ; Fuchs et al. 1993Citation ; Kachalsky et al. 1993, 1995Citation ) or (2) due to the glycosylation of the nAChR ligand binding domain that is absent in {alpha}-BTX-sensitive species (Kreienkamp et al. 1994Citation ; Keller et al. 1995Citation ). These proposals, however, (1) are not consistent with or cannot address the hypothesis that the {alpha}-BTX binding site is conserved across the species of Chordata, including the Elapidae snakes; (2) provide contradictory results regarding the role of amino acid residues forming the {alpha}-BTX binding site; and (3) have never been demonstrated using the ligand binding domain of the cobra (Naja spp.) or any other {alpha}-neurotoxin-resistant species as part of a functional nAChR.

In the present study, we tested the hypothesis that the {alpha}-neurotoxin binding site of the cobra (Naja spp.) nAChR is conserved per se and addressed the unsettled question of the molecular mechanism of resistance to conspecific {alpha}-neurotoxin. We cloned the Egyptian cobra (N. haje) nAChR ligand binding domain and expressed it as part of a functional nAChR in Xenopus oocytes. Using a two-microelectrode voltage clamp to monitor the ACh-induced currents, we tested the pharmacological action of {alpha}-BTX on nAChR containing the N. haje ligand binding domain and several subchimeric and point-mutated derivatives.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Cloning the N. haje Ligand Binding Domain and Construction of {alpha}N1
Total RNA isolated from the trunk skeletal muscle of the Egyptian cobra (N. haje, Elapidae, Reptilia) was reverse-transcribed with M-MLV reverse transcriptase using oligo (dT)-18 primer. The first-strand cDNA was PCR-amplified using gene-specific primers 5'-CACCTATTTCCCCTTTGATGAGCA-3' (sense) and 5'-ATGATGACGTTGACAATGAAGTAGAGA-3' (antisense; PA), followed by a second amplification with primers PA and 5'-TGAGCAAAACTGCAGTATGAAGCTGG-3' (sense). The final product was digested with HincII and PstI restriction enzymes (recognition sites are underlined) and ligated into the homologous position of the mouse (M. musculus) nAChR {alpha} subunit that was subcloned into a pSP64T vector (provided together with nAChR subunits ß, {gamma}, and {delta} by Arthur Karlin, Columbia University). The resulting chimeric subunit, {alpha}N1, was sequenced in both strands and was also used as a template to construct subsequent {alpha} subunit derivatives. All nucleotide and amino acid sequences are numbered according to the M. musculus nAChR {alpha} subunit sequence (GenBank accession number X03986).

Construction of Mutant {alpha} Subunits {alpha}N2–{alpha}N10
Segments or point mutations were introduced into wild-type {alpha} (to construct {alpha}N2, {alpha}N4, {alpha}N6, and {alpha}N8), {alpha}N1 (to construct {alpha}N3 and {alpha}N7), or {alpha}N3 (to construct {alpha}N5, {alpha}N9, and {alpha}N10) by specially designed PCR primers (within nucleotide positions 545–578) encoding the respective mutations. These special mutated primers, which also overlapped the unique restriction site DraIII-561, were used together with primers upstream of the EagI-295 site, 5'-CTTGAAATGGAATCCAGATGACTA-3' (sense; for {alpha}N2, {alpha}N3), or downstream of the BstXI-1215 site, 5'-AAACACAGCCAGCGTCCCGATGAG-3' (antisense; for all other mutants), to PCR amplify the mutated segment from templates {alpha} (to construct {alpha}N3, {alpha}N8, and {alpha}N9), {alpha}N1 (to construct {alpha}N2 and {alpha}N7), {alpha}N3 (to construct {alpha}N4, {alpha}N5, and {alpha}N6), or {alpha}N8 (to construct {alpha}N10). The PCR products were double-digested with DraIII and EagI or with DraIII and BstXI restriction enzymes, gel-purified, and ligated into {alpha}, {alpha}N1, or {alpha}N3 at the homologous position. All mutations were confirmed by sequencing both strands.

Expression of nAChR in Xenopus Oocytes
nAChR subunit {alpha} (BamHI), {alpha}N1–{alpha}N10 (BamHI), ß (XbaI), {gamma} (XbaI), and {delta} (BamHI) cDNAs were linearized with the restriction enzymes indicated. cRNAs were transcribed in vitro using the mMessage mMachine kit (Ambion, Austin, Tex.) according to the manufacturer's instructions. The resulting cRNAs were quantified by electrophoresis. Xenopus laevis were anesthetized with 0.15 % (w/v) 3-aminobenzoic acid ethyl ester, and ovarian lobes were removed and incubated in Ca2+-free ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES [pH 7.6]) containing 50 mg/ml collagenase B (Boehringer Mannheim, Indianapolis, Ind.) for 45 min at room temperature. Follicular layers were removed by forceps, and oocytes were transferred into ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES [pH 7.4]), supplemented with 2.5 mM Na-pyruvate, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2.5 % (v/v) fetal bovine serum, and incubated for 12 h before injection. cRNAs were mixed at a molar ratio of 2{alpha} : ß : {gamma} : {delta} (wild-type M. musculus) or 2{alpha}N1–{alpha}N10 : ß : {gamma} : {delta} (chimeric Naja/Mus {alpha}N1 and mutant {alpha}N2–{alpha}N10 subunits). Then 50 nl (200 pg/nl) of one of these mixtures was injected into the vegetal pole of the oocytes. Oocytes were incubated at 16°C and used for electrophysiological studies 24–72 h postinjection.

Acetylcholine (ACh) Dose-Response Recording
ACh-induced currents of the injected Xenopus oocytes were assayed with a two-microelectrode voltage clamp at a holding potential of -40 mV, using electrodes with <1.5 M{Omega} resistance when filled with 3.3 M KCl. ACh (Sigma, St. Louis, Mo.) was applied in the bath solution containing 115 mM NaCl, 2.5 mM KCl, 1.8 mM MgCl2, 1µM atropine, and 10 mM HEPES (pH 7.5) at a flow rate of 6 ml/min at room temperature. Five to ten minutes after impalement with the electrodes, oocytes were exposed to 4 x 10-5 M of ACh for 10 s, and peak current amplitude (Imax) was recorded as the maximum change of holding current. Thereafter, at 5-min intervals, oocytes were exposed to various concentrations of ACh, ranging from 1 x 10-7 M to 2 x 10-5 M, for 10 s, and peak current amplitude (Itest) was recorded as the maximum change of holding current. At the end of each protocol, the application of 4 x 10-5 M of ACh for 10 s was repeated to ensure reproducibility. Representative normalized ACh dose-response relationships were calculated by fitting the peak currents to the Hill equation, I = Imax/{1 + (Kapp/[ACh])n}. Each data point displayed along the curves is the mean of Itest/Imax ± SE recorded from two to five individual oocytes.

{alpha}-BTX Dose-Response Recording
Five to ten minutes after impalement with the electrodes, oocytes were exposed twice (5 min apart) to 10-5 M of ACh for 10 s, and the peak current amplitudes were averaged (Imax). Following these control recordings, the same oocytes were superfused with bath solution containing various concentrations of {alpha}-BTX (isolated from the banded krait, B. multicinctus, Elapidae, Reptilia; Mebs et al. 1972Citation ; Calbiochem-Novabiochem, San Diego, Calif.), ranging from 3.60 x 10-10 M to 3.60 x 10-7 M for 10 min, then exposed again twice (5 min apart) to 10-5 M of ACh for 10 s in the continuous presence of the {alpha}-BTX. The peak current amplitudes from the last two ACh exposures were averaged (Itest). Itest/Imax values were plotted as a function of {alpha}-BTX concentration. For nAChRs that were inhibited by {alpha}-BTX, representative normalized dose-response curves were calculated by fitting the peak currents to the Hill equation. For nAChRs that were resistant to {alpha}-BTX, mean Itest/Imax data points were fitted by a linear regression. Each data point displayed for both {alpha}-BTX-sensitive and {alpha}-BTX-resistant nAChRs along the curves is the mean of Itest/Imax ± SE recorded from 2–13 individual oocytes. Because of the slow onset of action of {alpha}-BTX, the time of incubation in this protocol does not allow measurements at equilibrium conditions. While not reaching equilibrium limits the precision of our measurements, it does not alter the conclusions from our experiments. Incubation of {alpha}N8 nAChR (single residue introduced into wild type, M. musculus {alpha}-BTX-sensitive nAChR) at the highest {alpha}-BTX concentration (3.6 x 10-7 M) tested for 30 min resulted in no current reduction in response to ACh.

Western Blot Analysis
Twenty-three to forty-eight Xenopus oocytes per cRNA samples were injected and incubated as described in the Expression of nAChR in Xenopus Oocytes section. After 2 days, oocytes were homogenized (20 µl/oocyte) in ice-cold 50 mM Na2HPO4/NaH2PO4 (pH 7.5), 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM benzamidine, 15 mM iodoacetamide, 2 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, and 5 µg/ml soybean trypsin inhibitor, then centrifuged at 15,000 x g for 20 min at 4°C. The pellet was resuspended in Laemmli sample buffer (15 µl/oocyte), incubated at 60°C for 20 min, and centrifuged at 15,000 x g at 4°C for 20 min. Electrophoresis was performed after the supernatant was loaded onto a 12% SDS-polyacrylamide gel. Kaleidoscope prestained standards (Bio-Rad, Hercules, Calif.) were used to monitor protein migration. After electrophoresis, gels were equilibrated in the transfer buffer (48 mM Tris base [pH 8.5], 192 mM glycine, 0.02% SDS, 20% methanol) for 30 min, then blotted for 3 h at 4°C onto NitroBind nitrocellulose membranes (Micron Separations, Westborough, Mass.) in a Bio-Rad transblot apparatus at 50 V in transfer buffer. Nonspecific binding sites were blocked by incubating the membrane in 50 mM Tris base (pH 7.5), 100 mM NaCl, 0.05 % (v/v) Tween 20 (TBS buffer) containing 5% (w/v) filtered nonfat dry milk (TBS-milk buffer) for 2 h at room temperature. Primary antibody, mAb210 (stock 1/94, 5 mg/ml IgG, provided by Jon Lindstrom, University of Pennsylvania), was diluted in TBS-milk buffer at 1:1,000 and incubated with the membrane for 2 h at room temperature, followed by washing for 30 min in TBS-milk buffer. Peroxidase-labeled affinity purified antibody to rat IgG produced in goat (Kirkegaard and Perry, Gaithersburg, Md.) was used as a secondary antibody. The anti-rat IgG antibody was diluted in TBS-milk buffer at 1:1,000 and incubated with the membrane for 1 h at room temperature. Membranes were then washed for 10 min in TBS-milk buffer, followed by 30 min in TBS buffer. The signal was detected by ECL Western blotting detection reagents (Amersham, Arlington Heights, Ill.) according to the manufacturer's instructions and recorded on Fuji medical RX film.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Cloning and Expression of the N. haje nAChR Ligand Binding Domain
To test the hypothesis that the {alpha}-neurotoxin binding site is conserved in Elapidae snakes (Elapidae, Reptilia), we cloned segment T148-P211 of the muscle-type nAChR {alpha} subunit from the Egyptian cobra (N. haje, Elapidae) (fig. 1 ). This segment contains several major residues implicated in the binding of the physiological ligand ACh and also contains the binding site for {alpha}-BTX in numerous other Chordata species representing various classes from Chondrichthyes to Mammalia (Karlin 1993Citation ). Specific residues of the muscle-type nAChR that are conserved across species and have been implicated in agonist binding by affinity-labeling include W149, Y151, Y190, C192, C193, and Y198 (Karlin 1993Citation ). These residues are conserved in the N. haje sequence.



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Fig. 1.—cDNA nucleotide (Naja, upper row) and deduced amino acid (Naja, middle row) sequence of the Egyptian cobra (Naja haje) nAChR {alpha} subunit ligand binding domain. For comparison, the homologous portion of the mouse (Mus musculus) amino acid (Mus, bottom row) sequence is also shown. Residues in M. musculus identical to N. haje are indicated with dashes. Nucleotide and amino acid sequences are numbered according to the M. musculus sequence (GenBank accession number X03986)

 
In order to pharmacologically test for the presence of the {alpha}-BTX binding site in the N. haje ligand binding domain, we constructed several chimeric and point-mutated nAChRs, termed {alpha}N1–{alpha}N10 (fig. 2 ). In these constructs, segments and/or single residues from N. haje or the chicken (Gallus gallus) were introduced into the homologous position of the mouse (M. musculus) nAChR {alpha} subunit, then coexpressed with the wild-type M. musculus ß, {gamma}, and {delta} subunits in Xenopus oocytes. All constructs were assayed for ACh-induced currents with two-microelectrode voltage clamp to monitor receptor function. The peak current amplitudes, individual time courses, and dose-response characteristics of ACh-induced responses were comparable with the wild-type M. musculus ({alpha}) in all chimeric and point-mutated ({alpha}N1–{alpha}N10) nAChRs (fig. 3 and table 1 ). These measurements indicated that any structural alterations in the nAChRs as a result of chimera construction or point mutation were minimal enough to maintain basic receptor functions.



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Fig. 2.—Sequences of the mutated {alpha} subunits that were constructed to test the {alpha}-BTX binding site of Naja haje nAChR. A, Schematic representation of a chimeric nAChR {alpha} subunit ({alpha}N1) indicating the introduction of the N. haje ligand binding domain (T154-L208) into the homologous position of the Mus musculus {alpha} subunit. B, Mutations of the {alpha} subunits introduced into the N. haje (upper panel) and M. musculus (lower panel) ligand binding domains. A dot indicates residues identical to the top sequence of each panel, {alpha}N1 or {alpha}. Shaded residues are from N. haje, and others are from M. musculus, except Y189 and A191, which are from the chicken (Gallus gallus). For simplicity, all nAChRs are referred to only by their respective {alpha} subunits in the text, figures, and table

 


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Fig. 3.—ACh-induced responses of wild-type and representative mutated nAChRs. A, Normalized ACh dose-response relationships of {alpha}, {alpha}N1, {alpha}N7, and {alpha}N8. B, Representative whole-cell currents induced by the application of 10-5 M ACh in {alpha}, {alpha}N1, {alpha}N7, and {alpha}N8

 

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Table 1 Physiological and Pharmacological Characteristics of the Mutated nAChRs

 
Mutational Analysis of the N. haje nAChR Ligand Binding Domain
Upon exposure to {alpha}-BTX, the wild-type ({alpha}) nAChR was inhibited in a dose-dependent manner with an IC50 of 3.50 x 10-9 M (fig. 4A and F ). However, nAChR containing the full-length N. haje ligand binding domain T154-L208 ({alpha}N1) was resistant (<10% inhibition) to the inhibitory action of 3.60 x 10-7 M {alpha}-BTX (fig. 4A and F ). Construction of two subchimeric nAChRs ({alpha}N2 and {alpha}N3) demonstrated that the {alpha}-BTX resistance-mediating elements were inherent in N. haje segment S187-L208 (fig. 4B ). Introduction of T154-H186 alone ({alpha}N2) did not alter the {alpha}-BTX sensitivity.



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Fig. 4.—Effect of {alpha}-BTX on the ACh-induced whole-cell currents on nAChR containing the Naja haje ligand binding domain and its mutated derivatives. Shown are normalized {alpha}-BTX dose-response relationships of (A) {alpha}N1; (B) {alpha}N2 and {alpha}N3; (C) {alpha}N4, {alpha}N5, and {alpha}N6; (D) {alpha}N7; and (E) {alpha}N8, {alpha}N9, and {alpha}N10. The dose-response relationship of the Mus musculus wild-type {alpha} is also shown for comparison (AE). F, Representative ACh-induced whole-cell currents of the nAChRs before (C) and after ({alpha}-BTX) superfusion with 3.6 x 10-8 M ({alpha}, {alpha}N7) or 3.6 x 10-7 M ({alpha}N1, {alpha}N8) {alpha}-BTX

 
In contrast to most {alpha}-BTX-sensitive species, positions 187 and 189 of the N. haje ligand binding domain contain nonaromatic residues. Lack of aromatic residues at these positions in Naja was claimed to eliminate the {alpha}-BTX binding site from the nAChR (McLane et al. 1991Citation ; Ohana et al. 1991Citation ; Barchan et al. 1992, 1995Citation ; Fuchs et al. 1993Citation ; Kachalsky et al. 1995Citation ). To address the role of these residues, we constructed three additional mutations ({alpha}N4, {alpha}N5, and {alpha}N6) in which aromatic amino acid residues present only in {alpha}-BTX sensitive species at positions 187 and 189 were introduced into N. haje segment S187-L208. The ACh-induced response of {alpha}N4 was resistant, while {alpha}N5 and {alpha}N6 were sensitive to the inhibitory action of {alpha}-BTX, with IC50 = 0.67 x 10-9 M and IC50 = 0.81 x 10-9 M, respectively (fig. 4C ). These results demonstrated that either N189 alone or the intact N-glycosylation signal (N189-X190-S191) was responsible for mediating resistance to {alpha}-BTX.

To differentiate between these two alternatives, a point mutation was designed that eliminated the N-glycosylation consensus sequence while leaving N189 intact. In addition to N189, this mutation also left all other residues (S187, L194, absence of parallel P194 and P197 pattern) that have been claimed to confer {alpha}-BTX resistance per se (McLane et al. 1991Citation ; Ohana et al. 1991Citation ; Barchan et al. 1992, 1995Citation ; Fuchs et al. 1993Citation ; Kachalsky et al. 1995Citation ) unaltered. S191 in the full N. haje ligand binding domain T154-L208 was mutated to alanine ({alpha}N7), a residue that is present in both {alpha}-BTX-sensitive (e.g., genus Xenopus, Gallus, Crocidura) {alpha}-BTX-resistant (e.g., Herpestes ichneumon) taxa. Western blot analysis was consistent with the lack of glycosylation in {alpha}N7 when compared with {alpha}N1 that had the intact N. haje ligand binding domain (fig. 5 ). This single point mutation of the N. haje ligand binding domain eliminated the resistance to {alpha}-BTX, yielding an IC50 of 9.11 x 10-9 M, comparable with the wild-type M. musculus IC50 of 3.50 x 10-9 M (fig. 4D and F ). A major consideration in this case is that all residues and motifs that have been claimed to confer {alpha}-BTX resistance per se (McLane et al. 1991Citation ; Ohana et al. 1991Citation ; Barchan et al. 1992, 1995Citation ; Fuchs et al. 1993Citation ; Kachalsky et al. 1995Citation ; Arias 2000Citation ) are present in {alpha}N7, including S187, N189, L194, and the absence of the parallel P194 and P197 pattern.



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Fig. 5.—nAChR {alpha} subunits carrying the consensus sequence for N-glycosylation exhibit slower electrophoretic mobility compared with {alpha} subunits lacking this signal. Shown is Western blot analysis of membrane proteins expressed in Xenopus oocytes after injection with water (none), or with cRNAs encoding the {alpha}, {alpha}N8, {alpha}N1, and {alpha}N7 nAChRs. The presence of the N-glycosylation consensus sequence is indicated by a plus sign, and its absence is indicated by a minus sign

 
Transferring the N. haje {alpha}-BTX Resistance to M. musculus nAChR
Single-residue mutations presented above were introduced into the N. haje sequence where they had no effect on {alpha}-BTX resistance or converted the N. haje ligand binding domain to {alpha}-BTX-sensitive. Can the N-glycosylation signal that is responsible for {alpha}-BTX resistance be transferred to a different species where it will confer its associated pharmacological effect observed in N. haje? We tested this transferability of {alpha}-BTX resistance and introduced the N. haje N-glycosylation signal into the wild-type M. musculus nAChR as a single point mutation, F189N ({alpha}N8), and as a double mutant, W187S/F189N ({alpha}N10). The W187S mutation was also introduced alone ({alpha}N9). Western blot analysis was consistent with the presence of glycosylation in {alpha}N8, with the M. musculus nAChR containing only one residue from N. haje (fig. 5 ). In agreement with the above findings, nAChRs {alpha}N8 and {alpha}N10 were resistant to 3.60 x 10-7 M {alpha}-BTX, while {alpha}N9, which lacked the consensus sequence for N-glycosylation, was inhibited with an IC50 of 7.51 x 10-9 M (fig. 4E ).


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
The present study demonstrated that the binding site for conspecific {alpha}-neurotoxin in Elapidae snakes is conserved in the nAChR per se and supports the hypothesis that animal toxins are targeted against evolutionarily conserved molecular motifs. In addition, we presented direct evidence that an animal species can be resistant to a component of conspecific venom by structural modification of the target molecule. These results also call for a revision of the binding site for {alpha}-BTX, one of the most widely utilized animal toxins.

Expression the Egyptian cobra (N. haje) ligand binding domain as part of a functional nAChR showed that the presence of the unique N-glycosylation signal in the middle of the ligand binding domain prevents the inhibitory action of {alpha}-BTX. The N-glycosylation signal, however, does not interfere with currents induced by the substantially smaller natural ligand ACh. Furthermore, the introduction of the N-glycosylation signal to the homologous position of mouse (M. musculus) nAChR also transfers the {alpha}-BTX resistance but has no effect on currents induced by ACh. Consistent with these observations, M. musculus nAChR carrying such N-glycosylation signal expressed in mammalian tissue culture exhibits greatly reduced affinity for {alpha}-BTX (Kreienkamp et al. 1994Citation ; Keller et al. 1995Citation ).

We demonstrated that the N-glycosylation is masking a genuine binding site for {alpha}-BTX on the nAChR. Several earlier studies based on synthetic peptides and bacterial fusion proteins suggested that the cobra (Naja spp.) has no binding site for {alpha}-BTX on the nAChR because of distinct amino acid substitutions in the polypeptide backbone per se, namely, S187 (McLane et al. 1991Citation ; Barchan et al. 1992, 1995Citation ; Fuchs et al. 1993Citation ; Kachalsky et al. 1995Citation ), N189 (McLane et al. 1991Citation ; Ohana et al. 1991Citation ; Barchan et al. 1992, 1995Citation ; Fuchs et al. 1993Citation ; Kachalsky et al. 1995Citation ), L194 (McLane et al. 1991Citation ; Barchan et al. 1992Citation ; Kachalsky et al. 1995Citation ), and/or lack of parallel P194 and P197 (Fuchs et al. 1993Citation ; Kachalsky et al. 1995Citation ). This proposal is in conflict with the hypothesis that the {alpha}-BTX binding site is conserved in Elapidae and inconsistent with results of the present experiments. When the N-glycosylation signal is removed by a single point mutation or multiple point mutations from the N. haje ligand binding domain, the nAChR retains its normal response to ACh, but a sensitivity to {alpha}-BTX that is comparable with mammalian nAChR is revealed (table 1 ). This inhibition demonstrates that the {alpha}-BTX binding site is in fact present in the Naja nAChR polypeptide backbone per se. While such conservation of the {alpha}-BTX binding site in Elapidae snakes that produce lethal {alpha}-neurotoxins is remarkable, it is not unexpected. The function of {alpha}-neurotoxins is to inhibit the nAChR in a phylogenetically wide spectrum of species. Therefore, residues forming the target site for {alpha}-BTX on the nAChR must be highly conserved in most lineages of Chordata and, consequently, likely to be essential for receptor structure and/or function in those species, including the Elapidae snakes. The requirement of the {alpha}-neurotoxin target site for nAChR physiology is probably explained by its overlap with or proximity to the binding site for the natural transmitter ACh (and possibly other domains critical for receptor physiology). Within the {alpha}-BTX binding segment E172-F205, there are several residues that are major determinants for ACh binding (Karlin 1993Citation ) and all of those residues are conserved among different taxa of Chordata. Evolutionary pressure to conserve the ACh binding site (and possibly other domains critical for receptor physiology) in Elapidae snakes, however, did not permit significant enough structural alteration in this segment of the nAChR polypeptide backbone, likely a major factor contributing to the conservation of the neurotoxin binding site.

The fact that the N. haje ligand binding domain is inhibited by {alpha}-BTX when the N-glycosylation signal is eliminated also calls for a revision of the current model of the {alpha}-BTX binding site. On the Chordata nAChR {alpha} subunit, residues W187 (Mulac-Jericevic and Atassi 1986Citation ; Neumann et al. 1986aCitation ; Gotti et al. 1988Citation ; Mulac-Jericevic et al. 1988Citation ; Fuchs et al. 1993Citation ; Spura et al. 1999, 2000)Citation , Y189 (Mulac-Jericevic and Atassi 1986Citation ; Neumann et al. 1986aCitation ; Gotti et al. 1988Citation ; Mulac-Jericevic et al. 1988Citation ; Conti-Tronconi et al. 1991Citation ; McLane et al. 1991Citation ; Ohana et al. 1991Citation ; Chaturvedi, Donnelly-Roberts, and Lentz 1992, 1993Citation ; McCormick et al. 1993Citation ; McLane, Wu, and Conti-Tronconi 1994Citation ; Levandoski et al. 1999Citation ) and other aromatic residues at positions 187 and/or 189 (Neumann et al. 1986aCitation ; Fuchs et al. 1993Citation ; Barchan et al. 1995Citation ; Kachalsky et al. 1995Citation ; Balass, Katchalski-Katzir, and Fuchs 1997Citation ; Spura et al. 1999, 2000Citation ), T189 (McCormick et al. 1993Citation ), adjacent aromatic residues at positions 189 and 190 (Conti-Tronconi et al. 1991Citation ; Chaturvedi, Donnelly-Roberts, and Lentz 1993Citation ; McLane, Wu, and Conti-Tronconi 1994Citation ; Balass, Katchalski-Katzir, and Fuchs 1997Citation ), T191 (Mulac-Jericevic and Atassi 1986Citation ; Neumann et al. 1986aCitation ; Gotti et al. 1988Citation ; Mulac-Jericevic et al. 1988Citation ), and P194 alone (Conti-Tronconi et al. 1991Citation ; Ohana et al. 1991Citation ; Chaturvedi, Donnelly-Roberts, and Lentz 1992, 1993Citation ; Fuchs et al. 1993Citation ; McCormick et al. 1993Citation ; Spura et al. 1999Citation ) or in parallel with P197 (Ohana and Gershoni 1990Citation ; McLane, Wu, and Conti-Tronconi 1994Citation ; Kachalsky et al. 1995Citation ) have been identified by others as being required for {alpha}-BTX binding based on synthetic peptides, bacterial fusion proteins, screening of phage-epitope libraries, and binding assays with cysteine-substituted mutants. However, we show here that the ligand binding domain of the N. haje nAChR lacks all of these residues or residue patterns but is still inhibited by {alpha}-BTX once the N-glycosylation signal is removed, indicating that these elements are not specifically required for {alpha}-BTX action. The discrepancies between these earlier findings by others and the present study are likely due to the lack of conformational forces (e.g., disulfide bridges, intersubunit contacts, effects of plasma membrane environment) in small peptides that are needed for proper protein folding in vivo, the presence of exogenous protein sequences, the lack of posttranslational modification, and the inability of in vitro binding assays to represent physiological receptor-ligand interaction. Furthermore, point mutations introduced into a short peptide sequence may have a more drastic effect than would be seen in the native protein that is subject to a large number of conformational constraints. In contrast, nAChRs functionally expressed in Xenopus oocytes exhibiting normal responses to ACh are not subject to such conformational limitations and should model native receptor characteristics with much higher fidelity. Consistent with our results, studies on {alpha}-BTX binding to nAChRs expressed in mammalian tissue cultures (Kreienkamp et al. 1994Citation ; Keller et al. 1995Citation ; Ackermann and Taylor 1997Citation ) or in Xenopus oocytes (Tomaselli et al. 1991Citation ) provided no support for the requirements of some of the residues identified by the various in vitro peptide or protein studies (Mulac-Jericevic and Atassi 1986Citation ; Neumann et al. 1986aCitation ; Gotti et al. 1988Citation ; Mulac-Jericevic et al. 1988Citation ; Ohana and Gershoni 1990Citation ; Conti-Tronconi et al. 1991Citation ; McLane et al. 1991Citation ; Ohana et al. 1991Citation ; Chaturvedi, Donnelly-Roberts, and Lentz 1992, 1993Citation ; Fuchs et al. 1993Citation ; McCormick et al. 1993Citation ; McLane, Wu, and Conti-Tronconi 1994Citation ; Barchan et al. 1995Citation ; Kachalsky et al. 1995Citation ; Balass, Katchalski-Katzir, and Fuchs 1997Citation ). Rather, V188, Y190, P197, and D200 were pointed out as binding determinants for another {alpha}-neurotoxin, Naja mossambica NmmI, to M. musculus nAChR (Ackermann and Taylor 1997Citation ). Since these residues are also present in the N. haje ligand binding domain, they further support the conservation of the {alpha}-neurotoxin binding site in Elapidae snakes.

The pharmacological data presented here, along with an analysis of Chordata nAChR protein sequences, provide evidence for a striking example of convergent evolution at the molecular level (fig. 6 ). The diet of the mongooses (Herpestes spp., Viverridae, Carnivora, Mammalia) includes cobras (Naja spp.), and they are the only nonreptilian Chordata species that are known to be naturally resistant to Elapidae venoms (Ovadia and Kochva 1997). The nAChR ligand binding domain of Herpestes ichneumon does in fact contain an N-glycosylation signal (Barchan et al. 1992Citation ), only two residues N-terminal (N187-X188-T189) from the position in which it is present in N. haje. The taxonomically closest species examined, the domestic cat (Felis catus, Felidae), another member of Carnivora (Barchan et al. 1995Citation ), as well as all other Chordata except advanced Squamata (Anguimorpha lizards and snakes, Reptilia), are {alpha}-BTX-sensitive (Burden, Hartzell, and Yoshikami 1975Citation ; Liu, Xu, and Hsu 1990Citation ; Endo and Tamiya 1991Citation ) and they all lack such a glycosylation signal in the ligand binding domain of the nAChR.



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Fig. 6.—Maximum-homology alignment of the protein sequences of nAChR ligand binding domains from various Chordata species. Boxed species are resistant to {alpha}-neurotoxins and contain an N-glycosylation signal in the ligand binding domain. Sequences are aligned in comparison with Naja haje. Dots indicate residues identical to those of N. haje; shaded residues are conserved in Chordata muscle-type nAChR {alpha} subunits

 
Based on the present work and theoretical considerations, we propose that the modified receptor structure resistance mechanism has a widespread occurrence among poisonous and venomous animals. For example, neurotoxins ATX II from the sea anemone Anemonia sulcata and BmK I from the scorpion Mesobuthus martensi share a common binding site on the voltage-gated Na+ channel (Catterall et al. 1980Citation ). Both of these toxins remain without effect in serum-free preparations of M. martensi abdominal nerve fibers (Terakawa et al. 1989Citation ). Similarly, tetrodotoxin (TTX) is another blocker of certain voltage-gated Na+ channels (Narahashi, Moore, and Scott 1964Citation ) that occurs in numerous species of animals, including the puffer fish, Tetraodontidae spp., and newts, Taricha spp. and Cynops spp. TTX is distributed in various tissues of the host species (Yotsu, Iorizzi, and Yasumoto 1990Citation ), and conspecific serum-free nerve fibers (Kao and Fuhrman 1967Citation ), retinal neurons (Kaneko, Matsumoto, and Hanyu 1997Citation ), and muscles (Kidokoro, Grinnell, and Eaton 1974Citation ) are insensitive to its action. Both of these examples suggest a modified receptor site for the toxins on the Na+ channel. The characters of the suggested structural modifications in these examples are unknown, but they can obviously occur by means other than N-glycosylation.

In summary, we demonstrated that the {alpha}-neurotoxin binding site on the nAChR of the Elapidae snakes is conserved. This genuine binding site is masked by an N-glycosylation signal that confers resistance against conspecific {alpha}-neurotoxin. The N. haje nAChR {alpha} subunit lacks residues W187, T189, Y189, other aromatic residues at positions 187 and 189, adjacent aromatic residues at positions 189 and 190, T191, and P194 alone or in parallel with P197; therefore, these structural motifs are not specifically required for {alpha}-BTX binding. The approach described here can be used to identify the mechanism of resistance against conspecific venoms in other species and to characterize toxin-receptor coevolution.


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 Abstract
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 Materials and Methods
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The sequence reported in this paper has been deposited in the GenBank database (accession number AF077763).


    Acknowledgements
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
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We thank Arthur Karlin (Columbia University), Robert S. Wilkinson (Washington University), Jon M. Lindstrom, and Rene Anand (University of Pennsylvania) for their suggestions and technical comments during the study, and we thank Arthur Karlin for his remarks on the manuscript.


    Footnotes
 
William Taylor, Reviewing Editor

1 Present address: Schering-Plough Research Institute, CNS-CV Research, Kenilworth, N.J. Back

1 Abbreviations: ACh, acetylcholine; {alpha}-BTX, {alpha}-bungarotoxin; N. haje, Egyptian cobra (Naja haje, Elapidae, Reptilia); nAChR, muscle-type nicotinic acetylcholine receptor. Back

2 Keywords: evolution snake neurotoxin resistance acetylcholine receptor Naja Elapidae Back

3 Address for correspondence and reprints: Zoltan Takacs, Laboratory of Evolutionary Genetics, Center for Environmental Research and Conservation, Columbia University, MC 5556, 1200 Amsterdam Avenue, New York, New York 10027. zoltan{at}zoltantakacs.com . Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 

    Ackermann E. J., P. Taylor, 1997 Nonidentity of the; ga-neurotoxin binding sites on the nicotinic acetylcholine receptor revealed by modification in {alpha}-neurotoxin and receptor structures Biochemistry 36:12836-12844[ISI][Medline]

    Arias H. R., 2000 Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors Neurochem. Intl 36:595-645[ISI][Medline]

    Balass M., E. Katchalski-Katzir, S. Fuchs, 1997 The {alpha}-bungarotoxin binding site on the nicotinic acetylcholine receptor: analysis using phage-epitope library Proc. Natl. Acad. Sci. USA 94:6054-6058[Abstract/Free Full Text]

    Barchan D., S. Kachalsy, D. Neumann, Z. Vogel, M. Ovadia, E. Kochva, S. Fuchs, 1992 How the mongoose can fight the snake: the binding site of the mongoose acetylcholine receptor Proc. Natl. Acad. Sci. USA 89:7717-7721[Abstract]

    Barchan D., M. Ovadia, E. Kochva, S. Fuchs, 1995 The binding site of the nicotinic acetylcholine receptor in animal species resistant to {alpha}-bungarotoxin Biochemistry 34:9172-9176[ISI][Medline]

    Barkas T., A. Mauron, B. Roth, C. Alliod, S. J. Tzartos, M. Ballivet, 1987 Mapping the main immunogenic region and toxin-binding site of the nicotinic acetylcholine receptor Science 235:77-80[ISI][Medline]

    Blount P., J. P. Merlie, 1989 Molecular basis of the two nonequivalent ligand binding sites of the muscle nicotinic acetylcholine receptor Neuron 3:349-357[ISI][Medline]

    Burden S. J., H. C. Hartzell, D. Yoshikami, 1975 Acetylcholine receptors at neuromuscular synapses: phylogenetic differences detected by snake {alpha}-neurotoxins Proc. Natl. Acad. Sci. USA 72:3245-3249[Abstract]

    Catterall W. A., 1980 Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes Annu. Rev. Pharmacol. Toxicol 20:15-43[ISI][Medline]

    Chaturvedi V., D. L. Donnelly-Roberts, T. L. Lentz, 1992 Substitution of Torpedo acetylcholine receptor {alpha}1-subunit residues with snake {alpha}1- and rat nerve {alpha}3-subunit residues in recombinant fusion proteins: effect on {alpha}-bungarotoxin binding Biochemistry 31:1370-1375[ISI][Medline]

    ———. 1993 Effects of mutations of Torpedo acetylcholine receptor {alpha}1 subunit residues 184–200 on {alpha}-bungarotoxin binding in a recombinant fusion protein Biochemistry 32:9570-9576[ISI][Medline]

    Conti-Tronconi B. M., B. M. Diethelm, X. D. Wu, F. Tang, T. Bertazzon, B. Schroder, S. Reinhardt-Maelicke, A. Maelicke, 1991 {alpha}-Bungarotoxin and the competing antibody WF6 interact with different amino acids within the same cholinergic subsite Biochemistry 30:2575-2584[ISI][Medline]

    Czajkowski C., A. Karlin, 1995 Structure of the nicotinic receptor acetylcholine-binding site. Identification of acidic residues in the {delta} subunit within 0.9 nm of the 5 {alpha} subunit-binding J. Biol. Chem 270:3160-3164[Abstract/Free Full Text]

    Endo T., N. Tamiya, 1991 Structure-function relationships of postsynaptic neurotoxins from snake venoms Pp. 165–222 in A. L. Harvey, ed. Snake toxins. Pergamon Press, New York

    Fuchs S., D. Barchan, S. Kachalsky, D. Neumann, M. Aladjem, Z. Vogel, M. Ovadia, E. Kochva, 1993 Molecular evolution of the binding site of the acetylcholine receptor Ann. N.Y. Acad. Sci 681:126-139[ISI][Medline]

    Gotti C., F. Frigerio, M. Bolognesi, R. Longhi, G. Racchetti, F. Clemeti, 1988 Nicotinic acetylcholine receptor: a structural model for {alpha}-subunit peptide 181–201, a putative binding site for cholinergic agents FEBS Lett 228:118-122[ISI][Medline]

    Kachalsky S. G., M. Aladjem, D. Barchan, S. Fuchs, 1993 The ligand binding domain of the nicotinic acetylcholine receptor. Immunological analysis FEBS Lett 318:264-268[ISI][Medline]

    Kachalsky S. G., B. S. Jensen, D. Barchan, S. Fuchs, 1995 Two subsites in the binding domain of the acetylcholine receptor: an aromatic subsite and a proline subsite Proc. Natl. Acad. Sci. USA 92:10801-10805[Abstract]

    Kaneko Y., G. Matsumoto, Y. Hanyu, 1997 TTX resistivity of Na+ channel in newt retinal neuron Biochem. Biophys. Res. Commun 240:651-656[ISI][Medline]

    Kao K. Y., F. A. Fuhrman, 1967 Differentiation of the actions of tetrodotoxin and saxitoxin Toxicon 5:25-34[ISI][Medline]

    Karlin A., 1993 Structure of nicotinic acetylcholine receptors Curr. Opin. Neurobiol 3:299-309[Medline]

    Keller S. H., H.-J. Kreienkamp, C. Kawanishi, P. Taylor, 1995 Molecular determinants conferring {alpha}-toxin resistance in recombinant DNA-derived acetylcholine receptors J. Biol. Chem 270:4165-4171[Abstract/Free Full Text]

    Kidokoro Y., A. D. Grinnell, D. C. Eaton, 1974 Tetrodotoxin sensitivity of muscle action potentials in pufferfishes and related fishes J. Comp. Physiol 89:59-72[ISI]

    Kreienkamp H.-J., S. M. Sine, R. K. Maeda, P. Taylor, 1994 Glycosylation sites selectively interfere with {alpha}-toxin binding to the nicotinic acetylcholine receptor J. Biol. Chem 269:8108-8114[Abstract/Free Full Text]

    Lentz T. L., 1995 Differential binding of nicotine and {alpha}-bungarotoxin to residues 173–204 of the nicotinic acetylcholine receptor {alpha}1 subunit Biochemistry 34:1316-1322[ISI][Medline]

    Levandoski M. M., Y. Lin, L. Moise, J. T. McLaughlin, E. Cooper, E. Hawrot, 1999 Chimeric analysis of a neuronal nicotinic acetylcholine receptor reveals amino acids conferring sensitivity to {alpha}-bungarotoxin J. Biol. Chem 274:26113-26119[Abstract/Free Full Text]

    Liu Y.-B., K. Xu, K. Hsu, 1990 Lack of the blocking effect of cobrotoxin from Naja naja atra venom on neuromuscular transmission in isolated nerve muscle preparations from poisonous and non-poisonous snakes Toxicon 28:1071-1076[ISI][Medline]

    McCormick D. J., J. A. Liebenow, G. E. Griesmann, V. A. Lennon, 1993 Nine residues influence the binding of {alpha}-bungarotoxin in {alpha}-subunit region 185–200 of human muscle acetylcholine receptor J. Neurochem 60:1906-1914[ISI][Medline]

    McLane K. E., X. Wu, B. M. Conti-Tronconi, 1994 An {alpha}-Bungarotoxin-binding sequence on the Torpedo nicotinic acetylcholine receptor {alpha}-subunit: conservative amino acid substitutions reveal side-chain specific interactions Biochemistry 33:2576-2585[ISI][Medline]

    McLane K. E., X. Wu, B. Diethelm, B. M. Conti-Tronconi, 1991 Structural determinants of {alpha}-bungarotoxin binding to the sequence segment 181–200 of the muscle nicotinic acetylcholine receptor {alpha} subunit: effects of cysteine/cysteine modification and species-species amino acid substitutions Biochemistry 30:4925-4934[ISI][Medline]

    Mebs D., K. Narita, S. Iwanaga, Y. Samejima, C.-Y. Lee, 1972 Purification, properties and amino acid sequence of {alpha}-bungarotoxin from the venom of Bungarus multicinctus Hoppe-Seyler Z. Physiol. Chem 353:243-262[ISI][Medline]

    Mulac-Jericevic B., M. Z. Atassi, 1986 Segment {alpha}182–198 of Torpedo californica acetylcholine receptor contains second toxin-binding region and binds anti-receptor antibodies FEBS Lett 199:68-74[ISI][Medline]

    Mulac-Jericevic B., T. Manshouri, T. Yokoi, M. Z. Atassi, 1988 The regions of {alpha}-neurotoxin binding on the extracellular part of the {alpha}-subunit of human acetylcholine receptor J. Protein Chem 7:173-177[ISI][Medline]

    Narahashi T., J. W. Moore, W. R. Scott, 1964 Tetrodotoxin blockage of sodium conductance increase in lobster giant axons J. Gen. Physiol 47:965-974[Abstract/Free Full Text]

    Neumann D., D. Barchan, M. Fridkin, S. Fuchs, 1986a. Analysis of ligand binding to the synthetic dodecapeptide 185–196 of the acetylcholine receptor {alpha} subunit Proc. Natl. Acad. Sci. USA 83:9250-9253[Abstract]

    Neumann D., D. Barchan, M. Horowitz, E. Kochva, S. Fuchs, 1989 Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteines of the {alpha} subunit Proc. Natl. Acad. Sci. USA 86:7255-7529[Abstract]

    Neumann D., D. Barchan, A. Safran, J. M. Gershoni, S. Fuchs, 1986b. Mapping of the {alpha}-bungarotoxin binding site within the {alpha} subunit of the acetylcholine receptor Proc. Natl. Acad. Sci. USA 83:3008-3011[Abstract]

    Ohana B., Y. Fraenkel, G. Navon, J. M. Gershoni, 1991 Molecular dissection of cholinergic binding sites: how snakes escape the effect of their own toxins? Biochem. Biophys. Res. Commun 179:648-654[ISI][Medline]

    Ohana B., J. M. Gershoni, 1990 Comparison of the toxin binding sites of the nicotinic acetylcholine receptor from Drosophila to human Biochemistry 29:6409-6415[ISI][Medline]

    Ovadia M., E. Kochva, 1977 Neutralization of Viperidae and Elapidae snake venoms by sera of different animals Toxicon 15:541-547[ISI][Medline]

    Pearce S. F., E. Hawrot, 1990 Intrinsic fluorescence of binding-site fragments of the nicotinic acetylcholine receptor: perturbations produced upon binding {alpha}-bungarotoxin Biochemistry 29:10649-10659[ISI][Medline]

    Sine S. M., T. Claudio, 1991 {gamma}- and {delta}-subunits regulate the affinity and the cooperativity of ligand binding to the acetylcholine receptor J. Biol. Chem 266:19369-19877[Abstract/Free Full Text]

    Spura A., R. U. Riel, N. D. Freedman, S. Agrawal, C. Seto, E. Hawrot, 2000 Biotinylation of substituted cysteines in the nicotinic acetylcholine receptor reveals distinct binding sites for {alpha}-bungarotoxin and erabutoxin a J. Biol. Chem 275:22452-22460[Abstract/Free Full Text]

    Spura A., T. S. Russin, N. D. Freedman, M. Grant, J. T. McLaughlin, E. Hawrot, 1999 Probing the agonist domain of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis reveals residues in proximity to the {alpha}-bungarotoxin binding site Biochemistry 38:4912-4921[ISI][Medline]

    Straight R., J. L. Glenn, C. C. Snyder, 1976 Antivenom activity of rattlesnake blood plasma Nature 261:259-260[ISI][Medline]

    Terakawa S., Y. Kimura, K. Hsu, Y.-H. Ji, 1989 Lack of effect of a neurotoxin from the scorpion Buthus martensi Karsch on nerve fibers of this scorpion Toxicon 27:569-578[ISI][Medline]

    Tomaselli G. F., J. T. McLaughlin, M. E. Jurman, E. Hawrot, G. Yellen, 1991 Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor Biophys. J 60:721-727[Abstract]

    Wilson P. T., T. L. Lentz, 1988 Binding of {alpha}-bungarotoxin to synthetic peptides corresponding to residues 173–204 of the {alpha} subunit of Torpedo, calf, and human acetylcholine receptor and restoration of high-affinity binding by sodium dodecyl sulfate Biochemistry 27:6667-6674[ISI][Medline]

    Wilson P. T., T. L. Lentz, E. Hawrot, 1985 Determination of the primary amino acid sequence specifying the {alpha}-bungarotoxin binding site on the {alpha} subunit of the acetylcholine receptor from Torpedo californica Proc. Natl. Acad. Sci. USA 82:8790-8794[Abstract]

    Yotsu M., M. Iorizzi, T. Yasumoto, 1990 Distribution of tetrodotoxin, 6-epitetrodotoxin, and 11-deoxytetrodotoxin in newts Toxicon 28:238-241[ISI][Medline]

Accepted for publication June 1, 2001.