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
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Abstract |
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Introduction |
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Venoms of the Elapidae snakes (e.g., cobras, kraits) contain several postsynaptic polypeptide -neurotoxins, such as
-bungarotoxin (
-BTX) isolated from the venom of the banded krait (Bungarus multicinctus) (Mebs et al. 1972
). The common target of
-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
2ß
(Karlin 1993
). Upon binding to the nAChR,
-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
-BTX on Chordata nAChR has been localized within segment E172-F205 of the
subunits (Wilson, Lentz, and Hawrot 1985
; Neumann et al. 1986a, 1986b
; Barkas et al. 1987
; Gotti et al. 1988
; Wilson and Lentz 1988
; Ohana and Gershoni 1990
; Pearce and Hawrot 1990
; Conti-Tronconi et al. 1991
; McLane et al. 1991
; Chaturvedi, Donnelly-Roberts, and Lentz 1992, 1993
; McLane, Wu, and Conti-Tronconi 1994
; Lentz 1995
; Arias 2000
). Specifically,
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
-BTX binding site in torpedo (Torpedo spp.), mouse (Mus musculus), and human (Mulac-Jericevic and Atassi 1986
; Neumann et al. 1986a
; Gotti et al. 1988
; Mulac-Jericevic et al. 1988
; Ohana and Gershoni 1990
; Conti-Tronconi et al. 1991
; McLane et al. 1991
; Ohana et al. 1991
; Barchan et al. 1992
; Chaturvedi, Donnelly-Roberts, and Lentz 1992
; Chaturvedi, Donnelly-Roberts, and Lentz 1993
; Fuchs et al. 1993
; McCormick et al. 1993
; McLane, Wu, and Conti-Tronconi 1994
; Kachalsky et al. 1995
; Lentz 1995
; Ackermann and Taylor 1997
; Spura et al. 1999, 2000
). 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
-BTX (Barchan et al. 1995
; Kachalsky et al. 1995
).
The primary binding site of -BTX, the
subunit segment E172-F205, is in the vicinity of
and
subunit interfaces (Blount and Merlie 1989
; Sine and Claudio 1991
; Czajkowski and Karlin 1995
). 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 1993
). 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 1976
). Such a neutralization mechanism has not been established in Elapidae snakes (Ovadia and Kochva 1977
), and serum-free neuromuscular preparations of snakes generally are insensitive to
-neurotoxins (Burden, Hartzell, and Yoshikami 1975
; Liu, Xu, and Hsu 1990
; Endo and Tamiya 1991
). Earlier studies suggested that
-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
subunit per se (Neumann et al. 1989
; McLane et al. 1991
; Ohana et al. 1991
; Barchan et al. 1992, 1995
; Chaturvedi, Donnelly-Roberts, and Lentz 1992
; Fuchs et al. 1993
; Kachalsky et al. 1993, 1995
) or (2) due to the glycosylation of the nAChR ligand binding domain that is absent in
-BTX-sensitive species (Kreienkamp et al. 1994
; Keller et al. 1995
). These proposals, however, (1) are not consistent with or cannot address the hypothesis that the
-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
-BTX binding site; and (3) have never been demonstrated using the ligand binding domain of the cobra (Naja spp.) or any other
-neurotoxin-resistant species as part of a functional nAChR.
In the present study, we tested the hypothesis that the -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
-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
-BTX on nAChR containing the N. haje ligand binding domain and several subchimeric and point-mutated derivatives.
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Materials and Methods |
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Construction of Mutant Subunits
N2
N10
Segments or point mutations were introduced into wild-type (to construct
N2,
N4,
N6, and
N8),
N1 (to construct
N3 and
N7), or
N3 (to construct
N5,
N9, and
N10) by specially designed PCR primers (within nucleotide positions 545578) 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
N2,
N3), or downstream of the BstXI-1215 site, 5'-AAACACAGCCAGCGTCCCGATGAG-3' (antisense; for all other mutants), to PCR amplify the mutated segment from templates
(to construct
N3,
N8, and
N9),
N1 (to construct
N2 and
N7),
N3 (to construct
N4,
N5, and
N6), or
N8 (to construct
N10). The PCR products were double-digested with DraIII and EagI or with DraIII and BstXI restriction enzymes, gel-purified, and ligated into
,
N1, or
N3 at the homologous position. All mutations were confirmed by sequencing both strands.
Expression of nAChR in Xenopus Oocytes
nAChR subunit (BamHI),
N1
N10 (BamHI), ß (XbaI),
(XbaI), and
(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
: ß :
:
(wild-type M. musculus) or 2
N1
N10 : ß :
:
(chimeric Naja/Mus
N1 and mutant
N2
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 2472 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 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.
-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 -BTX (isolated from the banded krait, B. multicinctus, Elapidae, Reptilia; Mebs et al. 1972
; 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
-BTX. The peak current amplitudes from the last two ACh exposures were averaged (Itest). Itest/Imax values were plotted as a function of
-BTX concentration. For nAChRs that were inhibited by
-BTX, representative normalized dose-response curves were calculated by fitting the peak currents to the Hill equation. For nAChRs that were resistant to
-BTX, mean Itest/Imax data points were fitted by a linear regression. Each data point displayed for both
-BTX-sensitive and
-BTX-resistant nAChRs along the curves is the mean of Itest/Imax ± SE recorded from 213 individual oocytes. Because of the slow onset of action of
-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
N8 nAChR (single residue introduced into wild type, M. musculus
-BTX-sensitive nAChR) at the highest
-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.
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Results |
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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 -BTX resistance per se (McLane et al. 1991
; Ohana et al. 1991
; Barchan et al. 1992, 1995
; Fuchs et al. 1993
; Kachalsky et al. 1995
) unaltered. S191 in the full N. haje ligand binding domain T154-L208 was mutated to alanine (
N7), a residue that is present in both
-BTX-sensitive (e.g., genus Xenopus, Gallus, Crocidura)
-BTX-resistant (e.g., Herpestes ichneumon) taxa. Western blot analysis was consistent with the lack of glycosylation in
N7 when compared with
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
-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
-BTX resistance per se (McLane et al. 1991
; Ohana et al. 1991
; Barchan et al. 1992, 1995
; Fuchs et al. 1993
; Kachalsky et al. 1995
; Arias 2000
) are present in
N7, including S187, N189, L194, and the absence of the parallel P194 and P197 pattern.
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Discussion |
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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 -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
-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
-BTX (Kreienkamp et al. 1994
; Keller et al. 1995
).
We demonstrated that the N-glycosylation is masking a genuine binding site for -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
-BTX on the nAChR because of distinct amino acid substitutions in the polypeptide backbone per se, namely, S187 (McLane et al. 1991
; Barchan et al. 1992, 1995
; Fuchs et al. 1993
; Kachalsky et al. 1995
), N189 (McLane et al. 1991
; Ohana et al. 1991
; Barchan et al. 1992, 1995
; Fuchs et al. 1993
; Kachalsky et al. 1995
), L194 (McLane et al. 1991
; Barchan et al. 1992
; Kachalsky et al. 1995
), and/or lack of parallel P194 and P197 (Fuchs et al. 1993
; Kachalsky et al. 1995
). This proposal is in conflict with the hypothesis that the
-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
-BTX that is comparable with mammalian nAChR is revealed (table 1
). This inhibition demonstrates that the
-BTX binding site is in fact present in the Naja nAChR polypeptide backbone per se. While such conservation of the
-BTX binding site in Elapidae snakes that produce lethal
-neurotoxins is remarkable, it is not unexpected. The function of
-neurotoxins is to inhibit the nAChR in a phylogenetically wide spectrum of species. Therefore, residues forming the target site for
-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
-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
-BTX binding segment E172-F205, there are several residues that are major determinants for ACh binding (Karlin 1993
) 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 -BTX when the N-glycosylation signal is eliminated also calls for a revision of the current model of the
-BTX binding site. On the Chordata nAChR
subunit, residues W187 (Mulac-Jericevic and Atassi 1986
; Neumann et al. 1986a
; Gotti et al. 1988
; Mulac-Jericevic et al. 1988
; Fuchs et al. 1993
; Spura et al. 1999, 2000)
, Y189 (Mulac-Jericevic and Atassi 1986
; Neumann et al. 1986a
; Gotti et al. 1988
; Mulac-Jericevic et al. 1988
; Conti-Tronconi et al. 1991
; McLane et al. 1991
; Ohana et al. 1991
; Chaturvedi, Donnelly-Roberts, and Lentz 1992, 1993
; McCormick et al. 1993
; McLane, Wu, and Conti-Tronconi 1994
; Levandoski et al. 1999
) and other aromatic residues at positions 187 and/or 189 (Neumann et al. 1986a
; Fuchs et al. 1993
; Barchan et al. 1995
; Kachalsky et al. 1995
; Balass, Katchalski-Katzir, and Fuchs 1997
; Spura et al. 1999, 2000
), T189 (McCormick et al. 1993
), adjacent aromatic residues at positions 189 and 190 (Conti-Tronconi et al. 1991
; Chaturvedi, Donnelly-Roberts, and Lentz 1993
; McLane, Wu, and Conti-Tronconi 1994
; Balass, Katchalski-Katzir, and Fuchs 1997
), T191 (Mulac-Jericevic and Atassi 1986
; Neumann et al. 1986a
; Gotti et al. 1988
; Mulac-Jericevic et al. 1988
), and P194 alone (Conti-Tronconi et al. 1991
; Ohana et al. 1991
; Chaturvedi, Donnelly-Roberts, and Lentz 1992, 1993
; Fuchs et al. 1993
; McCormick et al. 1993
; Spura et al. 1999
) or in parallel with P197 (Ohana and Gershoni 1990
; McLane, Wu, and Conti-Tronconi 1994
; Kachalsky et al. 1995
) have been identified by others as being required for
-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
-BTX once the N-glycosylation signal is removed, indicating that these elements are not specifically required for
-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
-BTX binding to nAChRs expressed in mammalian tissue cultures (Kreienkamp et al. 1994
; Keller et al. 1995
; Ackermann and Taylor 1997
) or in Xenopus oocytes (Tomaselli et al. 1991
) 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 1986
; Neumann et al. 1986a
; Gotti et al. 1988
; Mulac-Jericevic et al. 1988
; Ohana and Gershoni 1990
; Conti-Tronconi et al. 1991
; McLane et al. 1991
; Ohana et al. 1991
; Chaturvedi, Donnelly-Roberts, and Lentz 1992, 1993
; Fuchs et al. 1993
; McCormick et al. 1993
; McLane, Wu, and Conti-Tronconi 1994
; Barchan et al. 1995
; Kachalsky et al. 1995
; Balass, Katchalski-Katzir, and Fuchs 1997
). Rather, V188, Y190, P197, and D200 were pointed out as binding determinants for another
-neurotoxin, Naja mossambica NmmI, to M. musculus nAChR (Ackermann and Taylor 1997
). Since these residues are also present in the N. haje ligand binding domain, they further support the conservation of the
-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. 1992
), 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. 1995
), as well as all other Chordata except advanced Squamata (Anguimorpha lizards and snakes, Reptilia), are
-BTX-sensitive (Burden, Hartzell, and Yoshikami 1975
; Liu, Xu, and Hsu 1990
; Endo and Tamiya 1991
) and they all lack such a glycosylation signal in the ligand binding domain of the nAChR.
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In summary, we demonstrated that the -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
-neurotoxin. The N. haje nAChR
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
-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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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Present address: Schering-Plough Research Institute, CNS-CV Research, Kenilworth, N.J.
1 Abbreviations: ACh, acetylcholine; -BTX,
-bungarotoxin; N. haje, Egyptian cobra (Naja haje, Elapidae, Reptilia); nAChR, muscle-type nicotinic acetylcholine receptor.
2 Keywords: evolution
snake neurotoxin
resistance
acetylcholine receptor
Naja
Elapidae
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
.
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