We have characterized a unique toxin, Lqhb1, from the Old World scorpion, Leiurus quinquestriatus hebraeus, that resembles in sequence and activity both New World b-toxins as well as Old
Trang 1An ‘Old World’ scorpion b-toxin that recognizes both insect
and mammalian sodium channels
A possible link towards diversification of b-toxins
Dalia Gordon1, Nitza Ilan1,6, Noam Zilberberg2, Nicolas Gilles3, Daniel Urbach1, Lior Cohen1, Izhar Karbat1, Oren Froy1, Ariel Gaathon4, Roland G Kallen5, Morris Benveniste6and Michael Gurevitz1
1 Department of Plant Sciences, George S Wise Faculty of Life Sciences, Tel-Aviv University, Israel; 2 Department of Life Sciences, Ben-Gurion University, Israel; 3 CEA, De´partment d’Inge´nie´rie et d’Etudes des Prote´ines, France; 4 Bletterman Research Laboratory for Macromolecules, The Hebrew University-Hadassah, Medical School Jerusalem, Israel; 5 Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA;6Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Israel
Scorpion toxins that affect sodium channel (NaCh) gating
in excitable cells are divided into a-and b-classes Whereas
a-toxins have been found in scorpions throughout the world,
anti-mammalian b-toxins have been assigned, thus far, to
New World scorpions while anti-insect selective b-toxins
(depressant and excitatory) have been described only in the
Old World
2 This distribution suggested that diversification
of b-toxins into distinct pharmacological groups occurred
after the separation of the continents, 150 million years ago
We have characterized a unique toxin, Lqhb1, from the Old
World scorpion, Leiurus quinquestriatus hebraeus, that
resembles in sequence and activity both New World
b-toxins as well as Old World depressant toxins Lqhb1
competes, with apparent high affinity, with anti-insect and
anti-mammalian b-toxins for binding to cockroach and rat
brain synaptosomes, respectively Surprisingly, Lqhb1 also
competes with an anti-mammalian a-toxin on binding to rat brain NaChs Analysis of Lqhb1 effects on rat brain and Drosophila Para NaChs expressed in Xenopus oocytes revealed a shift in the voltage-dependence of activation to more negative membrane potentials and a reduction in sodium peak currents in a manner typifying b-toxin activity Moreover, Lqhb1 resembles b-toxins by having a weak effect
on cardiac NaChs and a marked effect on rat brain and skeletal muscle NaChs
that Lqhb1 may represent an ancestral b-toxin group in Old World scorpions that gave rise, after the separation of the continents, to depressant toxins in Old World scorpions and
to various b-toxin subgroups in New World scorpions Keywords: scorpion toxins; sodium channel subtypes; toxin diversification
Scorpion long chain toxins affecting voltage-gated
sodium channels (NaCh) are polypeptides of 61–76 amino
acids long that traditionally are divided between two
major classes, a and b, according to their physiological
effects on channel gating and their binding properties
[1–3] a-Toxins slow sodium channel inactivation upon
binding to a homologous cluster of binding sites named
receptor site-3, and are subdivided into distinct groups
according to their potency for mammalian and insect
receptors and their affinity for sodium channel subtypes
[2,4–7] a-Toxins predominate in the venom of Buthidae
scorpions of the Old World (Africa and Asia), but some representatives have been also identified in New World (America) scorpions [1] b-Toxins shift the activation voltage of sodium channels to more negative membrane potentials upon binding to receptor site-4 [2,7,8], and vary greatly in their effects on various animals Css2 and Css4 (from Centruroides suffusus suffusus) show specificity for mammals [1]; Cll1 (from C limpidus limpidus), Cn5, and Cn11 (from C noxius) are highly effective on crustaceans [9–12]; Ts7 and Tst1 (from Tityus serrulatus and T stig-murus), and Tbs1 and Tbs2 (from T bahiensis) are highly effective on both insects and mammals [1,12–16] b-Toxins, active on mammals, have thus far been assigned
to scorpions of the New World (Tityus and Centruroides species), whereas depressant and excitatory b-toxins, which modify exclusively the activation of insect sodium channels, have been found strictly in Old World Buthoids [1,2,12,17,18]
In addition to their effects on sodium channel gating, classification of toxins to either the a-or b- class relies on competition binding assays against the Old World toxin, Aah2, from Androctonus australis hector, and the New World toxin, Css2, from Centruroides suffusus suffusus, respectively [1,2] Further distinction between toxins can be made using competition binding assays utilizing LqhaIT
Correspondence to D Gordon or M Gurevitz, Department of Plant
Sciences, George S Wise Faculty of Life Sciences,
Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel.
1
Fax: + 972 3 6406100, Tel.: + 972 3 6409844,
E-mail: dgordon@post.tau.ac.il or mamgur@post.tau.ac.il
Abbreviations: Lqhb1, Leiurus quinquestriatus hebraeus beta toxin 1;
LqhIT2, anti-insect selective depressant toxin; LqhaIT, anti-insect
a-toxin; Lqh2, anti-mammalian a-toxin; Css2, Css4, Centruroides
suffusus suffusus b-toxins 2 and 4; Ts7, Tityus serrulatus toxin 7
(also called Ts1 and c-toxin); NaCh, sodium channel.
(Received 27 March 2003, accepted 29 April 2003)
Trang 2toxins in the Old World occurred from an unknown
ancestral progenitor after the separation of the continents
150 million years ago
Yet, an Old World scorpion toxin, AahIT4, with high
affinity for the AahIT binding site on insect neuronal
membranes, could also compete with a-(Aah2) and
b-(Css2) anti-mammalian toxins for binding to rat brain
synaptosomes [23] As AahIT4 shares little sequence
similarity with any of the known anti-mammalian scorpion
toxins [1,12], and no information was available on its mode
of action, it was considered a unique member of a new
pharmacological group of neither a-nor b-type toxins
[12,23] More recently, two toxins have been purified from
the Asian scorpion, Buthus martensii Karsch, BmK AS and
BmK AS-1, which share 80 and 86% sequence identity
with AahIT4 [24] These toxins are weakly toxic to insects,
are not toxic to mice and inhibit Na+currents in neurons
of rat dorsal root ganglia [25] Still, no pharmacological
details that allow their classification to a-or b-toxins have
been provided
Here we report the isolation and characterization of an
Old World toxin, Lqhb1 that probably belongs to the same
group as AahIT4 if sequence similarity and binding features
are examined
5 Lqhb1 competes with both a-and b-toxins
for binding to rat brain synaptosomes and with excitatory
anti-insect selective toxins in insect neuronal membranes
We show that Lqhb1 affects insect and mammalian NaCh
subtypes in a manner that typifies New World b-toxins
This suggests that b-toxins affecting mammalian NaChs
have existed and are still present in Old World scorpions
The effects of Lqhb1 on various NaCh subtypes may
suggest that this toxin represents an ancient group of
b-toxins that gave rise to the anti-insect depressant toxins in
the Old World and to the b-toxins active on mammals in
the New World
Experimental procedures
Biological material
Venom from Leiurus quinquestriatus hebraeus was collected
from scorpion stings to a parafilm membrane Sarcophaga
falculata(blowfly) larvae and Periplaneta americana
(cock-roaches) were bred in the laboratory Albino laboratory
ICR mice were purchased from the Levenstein farm in
Yokneam, Israel As purified Lqhb1 was obtained in a
limited amount, the toxin was also purchased from Latoxan
(LTx-003; Valance, France) together with the
anti-mam-malian a-toxin, Lqh2
ammonium acetate that were toxic to blowfly larvae were combined and further purified via HPLC using an analytical
C18 column (250· 10 mm; Vydac, USA) Sample was loaded in 0.1% trifluoroacetic acid in water (Buffer A) and eluted with a stepwise increasing gradient of 0.1% trifluoro-acetic acid in acetonitrile (Buffer B) at a flow rate of
1 mLÆmin)1 The protein eluted after 19.5 min and con-tained 163 lg pure polypeptide of 7463 Da (determined by Electrospray Mass Spectrometry, Technion, Haifa, Israel) and exerted depressant activity on blowfly larvae Amino acid sequence analysis, carried out by automated Edman degradation using an Applied Biosystem (Foster City, CA, USA) gas-phase sequencer (470 A) connected to its corres-ponding PTH-analyzer (120 A) and data system (900 A) identified the first 24 N-terminal residues This amino acid sequence was used to pull out the entire cDNA clone using
back-to-back oligonucleotide primers in a PCR technique described by Zilberberg and Gurevitz [
degenerate oligonucleotides, designed according to the protein sequence (Primer 1: 5¢-ANACYTTPCANCC HGTNGC-3¢; Primer 2: 5¢-GGTGYGTNATHGAYGA YGC-3¢; N stands for either A, G, T or C; Y for C or T;
P for A or G; H for A, T, or C; Fig 1A), were used as primers for PCR (MJ Research thermocycler, USA) with
L q hebraeus cDNA library [27] as DNA template to amplify the entire Lqhb1-cDNA Reaction conditions were:
30 cycles of 1 min at 94C, 1 min 50 C and 1 min at
72C The PCR product was blunt-ended, phosphorylated, cloned into the SmaI site of pBluescript, and subjected to sequence analysis using Sequenase II (United States Biochemicals) The cloned gene was labeled with32P and used as a probe to pull out by colony hybridization the original cDNA from the library (Fig 1)
Recombinant toxin production Bj-xtrIT and LqhIT2 were produced in Escherichia coli strain BL21 and reconstituted by in vitro folding as was described previously [19,28] A synthetic gene was used to produce Css4 b-toxin (I Karbat, D Gordon & M Gurevitz, unpublished observation)
for LqhIT2 [28]
Toxicity assays Five toxin concentrations were tested using four-day-old blowfly larvae ( 100 mg body weight) Ten larvae were injected intersegmentally at the rear side with each toxin concentration in three independent experiments A positive
Trang 3result was scored when a characteristic paralysis (transient
immobilization and contraction replaced by gradually
increasing flaccidity) was obtained and lasted at least
15 min ED50 values were calculated as was described
previously [28]
Binding experiments
Insect synaptosomes were prepared from whole heads of
adult P americana according to a previously described
method [19] Mammalian brain synaptosomes were
pre-pared from adult albino Sprague–Dawley rats ( 300 g,
laboratory bred), as was described previously
Mem-brane protein concentration was determined using a
Bio-Rad Protein Assay, using bovine serum albumin (BSA) as a
standard Lqh2, LqhaIT, Bj-xtrIT and Css4 were
radio-iodinated by iodogen (Pierce, Rockford, USA) using 5 lg
toxin and 0.5 mCi carrier-free Na125I (Amersham, UK) and
the monoiodotoxins were purified using an analytical Vydac
RP-HPLC C18column, as was described previously [4,30]
The concentration of the radiolabeled toxin was determined
according to the specific activity of the125I corresponding to
2500–3000 d.p.m.Æfmol)1of monoiodotoxin, depending on
the age of the radiotoxin and by estimation of its biological
activity (usually 50–70%; see [30] for details) The
compo-sition of the medium used in the binding assays was (in mM):
choline Cl, 130; CaCl2, 1.8; KCl, 5; MgSO4, 0.8, Hepes 50;
Glucose 10, and 2 mgÆmL)1 BSA, pH 7 Wash buffer
composition was (in mM): choline Cl, 140; CaCl2, 1.8; KCl,
5.4; MgSO4, 0.8; Hepes, 50; 5 mgÆmL)1 BSA, pH 7.5
Binding assays were performed as was described previously
[29,30] Nonspecific toxin binding was determined in the
presence of a high concentration of unlabeled toxin, as
specified in figure legends, and consisted typically of 5–15%
of total binding Equilibrium competition binding assays were performed using increasing concentrations of the unlabeled toxins in the presence of a constant low con-centration of [125I]toxins, and analyzed by the computer programKALEIDAGRAPH(Synergy Software, Reading, PA, USA) using a nonlinear fit to the Hill equation (for IC50 determination) The Ki were calculated by the equation
Ki¼ IC50/[1 + (L*/Kd)], where L* is the concentration of hot toxin and Kdis its dissociation constant Each experiment was performed in duplicate samples and repeated multiple times as indicated (n) for each Kivalue Bj-xtrIT excitatory toxin, a marker of receptor site-4 in insect sodium channels [19,21], was used in competition binding assays of LqhIT2 and Lqhb1 toxins to cockroach neuronal membranes
Sodium channel expression and two-electrode voltage-clamp assays usingXenopus oocytes cRNAs encoding rat skeletal muscle (Nav1.4; rSkM1), rat brain IIa (rNav1.2a; rBIIA), human heart (hNav1.5; hH1) subtypes and insect Drosophila Para (DmNav1; gift from
J Warmke, Merck, New Jersey, USA)
a-subunits, and the auxiliary human b1 and insect TipE subunits (gift from M S Williamson, IACR-Rothamsted,
10
UK), were transcribed in vitro using T7 RNA polymerase and the mMESSAGE mMACHINETMsystem (Ambion, USA [31,32]); and were injected into Xenopus laevis oocytes
as described by Shichor et al [21] One to four days after injection, currents were measured by two-electrode voltage clamp using a Gene Clamp 500 amplifier (Axon Instru-ments, Union City, CA, USA) Data were sampled at
10 kHz and filtered at 5 kHz Data acquisition was con-trolled by a Macintosh PPC 7100/80 computer, equipped with ITC-16 analog/digital converter (Instrutech Corp.,
Fig 1 Nucleotide sequence of the cDNA clone
encoding Lqhb1 and its deduced amino acid
sequence The putative signal sequence is
underlined and a polyadenylation signal
appears in lowercase letters The amino acid
stretch used for design of back-to-back
oligo-nucleotide primers is indicated by arrows 1
and 2 (see Experimental procedures for
sequence) The technique used for cloning
has been described previously [26].
Trang 4Port Washington, NY, USA), utilizingSYNAPSE(Synergistic
Systems, Sweden) Capacitance transients and leak currents
were removed by subtracting a scaled control trace utilizing a
P/6protocol [32] Bath solution contained (in mM): NaCl, 96;
KCl, 2; MgCl2, 1; CaCl2, 1.8; Hepes, 5; pH 7.85 Toxins were
diluted with bath solution containing 1 mgÆmL)1 BSA
Oocytes were washed with bath solution flowing from a
BPS-8 perfusion system (ALA Scientific Instruments, Westbury,
NY, USA) with a positive pressure of 4 psi Approximately
1 mL of toxin-containing solution was perfused over the
oocyte situated in a 200-lL chamber at room temperature
Results
Purification and cloning of a functionally unique
b-toxin fromL q hebraeus
Lqhb1 was purified from the venom of L q hebraeus by
cation-exchange chromatography followed by RP-HPLC
(see Experimental procedures for details) Upon injection of Lqhb1 to blowfly larvae, a short, transient contraction was observed followed by a dose-dependent, long-lasting flaccid paralysis (ED50¼ 102 ng per 100 mg body weight) These effects on blowfly larvae are typical of depressant toxins, such as LqhIT2 [20,28] The amino acid sequence deduced from the cDNA nucleotide sequence reveals 73% sequence identity with AahIT4 [23], and 85 and 91% with BmK AS and BmK AS-1 [24], respectively (Fig 2) The relative molecular mass of Lqhb1, determined by mass spectroscopy,
is 7463 Da, which matches the calculated value of the amino acid sequence deduced from the cDNA nucleotide sequence This suggests that Lqhb1 does not undergo post-trans-lational processing as has been shown for other toxins [27] Binding of Lqhb1 to insect and rat brain NaCh
The sequence similarity between Lqhb1 and AahIT4 [23] could suggest similar activity and therefore we analyzed the
Fig 2 Sequence alignment of toxin representatives of various pharmacological groups that affect sodium channels The alignment relies on known structures, putative models [2] and conserved cysteine residues Dashes indicate gaps Asterisks (*) designate alpha-amidation of C-termini Cysteine residues that are conserved in all scorpion toxins and form disulfide bonds (plane lines) are shaded by light grey, whereas cysteines involved in unique disulfide bonds (dashed lines) are shaded by dark grey Aah, Androctonus australis hector; Bj, Buthotus judaicus; BmK, Buthus martensii Karsch; Cn, Centruroides noxius; Cll, Centruroides limpidus limpidus; CsE, Centruroides sculpturatus Ewing; Css, Centruroides suffusus suffusus; Lqh, Leiurus quinquestriatus hebraeus; Lqq, Leiurus quinquestriatus quinquestriatus; Ts, Tityus serrulatus; Tst, Tityus stigmurus; Tb, Tityus bahiensis [12].
Trang 5pharmacological profile of Lqhb1 in competition binding
assays utilizing well established a-and b-toxin markers
Lqhb1 inhibited, though at high concentration, the binding
of the classical anti-mammalian a-toxin, Lqh2 [5,29], to
site-3 in rat brain synaptosomes (Ki¼ 1.85 lM, n¼ 2;
Fig 3A) A significantly lower concentration of AahIT4
(60 nM) inhibited the binding of Aah2 [23] Lqhb1 also
competed with the anti-mammalian b-toxin, Css4, for
binding to site-4 in rat brain synaptosomes with very high
apparent affinity [Ki¼ 0.51 ± 0.27 nM, (mean ± SD),
n¼ 3; Fig 3B] In comparison, the apparent affinity of
AahIT4 for the binding site of Css2 is much lower
(IC50¼ 40 nM[23]) Although Lqhb1 competed with both
site-3 and site-4 toxins for binding to rat brain sodium
channels, it did not inhibit the binding of the a-insect toxin,
LqhaIT, to receptor site-3 on cockroach channels (not
shown) Yet, Lqhb1 inhibited the binding of the excitatory
toxin, Bj-xtrIT, to cockroach synaptosomes with high
apparent affinity (Ki¼ 0.170 ± 0.017 nM, n¼ 3; Fig 3C)
These results demonstrate the similarity in binding
capabi-lities between Lqhb1 and AahIT4, although they vary
quantitatively in the apparent affinity for receptor sites 3
and 4 in rat brain sodium channels
Effects of Lqhb1 on sodium channel subtypes
The effects of Lqhb1 on sodium currents were examined on
various mammalian and insect sodium channels expressed
in Xenopus oocytes using two-electrode voltage-clamp In
the absence of toxin, a step-depolarization from a holding
potential of )80 to )40 mV elicits almost no current in
oocytes expressing the rNav1.2a channels (Fig 4A)
How-ever, in the presence of 2.5 lM Lqhb1, a significant peak
current is observed (Fig 4A) that indicates a shift of
channel activation to more negative membrane potentials as
readily observed in Fig 4B In contrast, upon depolarization
to )20 mV, under which channel activation in the
absence of toxin was near maximal, peak currents were inhibited 70 ± 10% (n¼ 4) in the presence of Lqhb1 (Fig 4A,B) The current–voltage (I–V) relationship delin-eated in Fig 4B indicates two clear effects imposed by Lqhb1 on rNav1.2a channels The shift in the voltage-dependence of channel activation to more negative mem-brane potentials, and a marked decrease in the sodium peak-current amplitude typify the phenotypic change of sodium currents induced by scorpion b-toxins active on mammals, e.g Ts7 and Css4 [8,14,33–35] As New World b-toxins that affect mammals show weak activity on cardiac sodium channels [34,36], the specificity of Lqhb1 was further examined on rat skeletal muscle (rNav1.4) and human heart (hNav1.5) channels The toxin effects on rNav1.4 were similar to those obtained with rNav1.2a, whereas no shift in channel activation and only little decrease in peak current were observed in hNav1.5 (Fig 4C,D) These results indicate that Lqhb1 is similar
to other b-toxins in action and specificity to mammalian sodium channel subtypes
Lqhb1 is similar to the anti-insect depressant toxin, LqhIT2, in its toxic symptoms induced in blowfly larvae, and the ability to compete for the binding site of the excitatory toxin, Bj-xtrIT, in insect NaChs [19,22] There-fore, the electrophysiological effects of Lqhb1 and LqhIT2 were compared on an insect NaCh Both toxins revealed typical b-toxin effects on oocytes expressing the Drosophila Para NaCh and TipE The peak sodium current, elicited
by depolarization to )10 mV, decreased 45 ± 13% and
66 ± 5% (n¼ 3) in the presence of 2.5 lM Lqhb1 and LqhIT2, respectively In addition, the I–V curves obtained indicate that the appearance of the sodium current is shifted
to more negative potentials in the presence of either toxin (Fig 5A) Similar effects were observed when LqhIT2 was applied on an isolated cockroach axon [18] However, in contrast to Lqhb1 (Fig 4B), LqhIT2 in concentrations as high as 50 lMdid not affect the rNa1.4 channel (Fig 5B),
Fig 3 Binding of Lqhb1 to receptor sites 3 and 4 in rat brain and cockroach sodium channels Competition of Lqhb1 with the site-3 a-toxin, Lqh2 (A), and with the site-4 b-toxin, Css4 (B), for binding to rat brain NaChs Rat brain synaptosomes (65 lg proteinÆmL)1) were incubated 30 min at 22 C with 110 p M [ 125 I]Lqh2 or 120 p M [ 125 I]Css4 and increasing concentrations of the indicated toxins Nonspecific binding, determined in the presence
of 200 n M Lqh2 or 1 l M Css4, respectively, was subtracted (C) Competition of Lqhb1 with [ 125 I]Bj-xtrIT for binding to cockroach NaCh Cockroach neuronal membranes (16 lgÆmL)1) were incubated for 60 min at 22 C with 180 p M [125I]Bj-xtrIT and increasing concentrations of the indicated toxins Nonspecific binding, determined in the presence of 1 l M Bj-xtrIT, was subtracted The amount of bound [125I]toxin is provided as the percentage of maximal specific binding without competitor The competition curves were analyzed using the nonlinear fit of the Hill equation (with a Hill coefficient of 1) for determining of the IC 50 values (see Experimental procedures) The data points are means of two to three measurements from representative experiments, of which the following K i values were obtained (in n M ): Lqh2, 2.1; Lqhb1, 1900 (in A) Css4, 1.1; Lqhb1, 0.64 (in B) Bj-xtrIT, 0.35; Lqhb1, 0.2 (in C).
Trang 6Jurassic Brazilo-Ethiopian continent (Africa–South Amer-ica) Scorpion a-toxins, that affect NaCh inactivation and resemble Old World classical a-toxins in their amino acid sequences, have been described in the New World, e.g CsE V, Ts IV, and TsTX V [1,12], which suggests that they existed before the separation of the continents
As b-toxins active on mammals have not been found thus far in scorpions of the Old World, it could be assumed that they have developed in Tityus and Centruroides New World scorpions after the separation of the continents [12] Yet, scorpion polypeptides that resemble New World b-toxins have been reported in the Old World and include the nontoxic polypeptide, AahSTR1 [38], the glycosylated toxin, Aah6 [39], and the anti-insect selective excitatory and depressant toxin groups [2,12,17,20] In addition, polypep-tides with some b-toxin properties have been found in the venom of the old world scorpions, Leiurus quinquestriatus hebraeus [40] and Buthus martensii Karsch [25] Another peculiar toxin that competes for both a and b-toxin receptor sites in rat brain synaptosomes, AahIT4, seems to be related
to b-toxins because it was recognized by antibodies raised against the b-toxin, Css2, but not by antibodies against the a-toxin, Aah2, or the excitatory toxin, AahIT [23] The pharmacological properties of Lqhb1 describe for the first time a typical b- toxin in the Old World The high similarity in sequence of Lqhb1, Bmk AS, BmK AS-1, and AahIT4 suggests that these toxins may belong to a unique group of Old World b-toxins (Fig 2) Lqhb1 resembles AahIT4 in its high apparent affinity for receptor site-4 in insect sodium channels (Fig 3 [23]) Yet, whereas AahIT4 has moderate affinity for both receptor sites 3 and 4 on rat brain synaptosomes [23], Lqhb1 competes with Lqh2 on binding to receptor site-3 only at high concentrations, and binds receptor site-4 in mammalian sodium channels with a very high apparent affinity (Fig 3) Although both toxins seem to belong to one pharmacological group, Lqhb1 exerts typical b-toxin binding properties to a greater extent than AahIT4 Lqhb1 and AahIT4 vary also in their effect on blowfly larvae as AahIT4 induces contraction [23] and Lqhb1 induces flaccid paralysis
The sequence of Lqhb1 resembles those of New World b-toxins (41–50% identity; Fig 2) The pharmacological features of Lqhb1 are mostly similar to those of the b-toxin, Ts7, with high affinity binding for insect and mammalian NaChs (Figs 3–5 [1,2,13]), and preference for mammalian brain and skeletal muscle NaCh subtypes (Fig 4 [34]) Notably, the sequence, binding, and electrophysiological properties of Lqhb1 show substantial resemblance to those
of Old World anti-insect selective depressant toxins, such
Fig 5 Comparison of the effects of Lqhb1 and LqhIT2 on insect sodium
channels Curves I–V were obtained from two-electrode voltage-clamp
experiments using Xenopus oocytes that coexpress the Drosophila Para
a-subunit with the auxiliary insect b-subunit TipE (A), or the rNa v 1.4
a-subunit with b1 (B) Peak amplitudes obtained in the absence of
toxin are designated by filled circles; and peak amplitudes measured
from traces elicited in the presence of toxin are represented by open
circles Lqhb1 or LqhIT2 (2.5 l M ) enabled Para channel sodium
cur-rents to be elicited at more hyperpolarized potentials, and decreased
the peak inward currents at potentials > )20 mV In contrast, 50 l M
LqhIT2 had no effect on currents mediated by rNa 1.4.
Fig 4 Effects of Lqhb1 on mammalian sodium channel subtypes.
Current–voltage (I–V) curves were obtained with two-electrode
voltage-clamp experiments using Xenopus oocytes that coexpress rat
brain, rNa v 1.2a (A, B), rat skeletal muscle, rNa v 1.4 (C), and human
heart, hNa v 1.5 (D) channel a-subunits together with the mammalian
auxiliary subunit b1 In A, currents elicited during a depolarizing pulse
to )40 mV (upper traces) or )20 mV (lower traces) in the absence and
presence of 2.5 l M Lqhb1, are shown Note that in the presence of
Lqhb1, the current appears during the )40 mV pulse, in contrast to the
control trace (B–D) Representative I–V curves are shown for
experi-ments in which the different channels were expressed in Xenopus
oocytes in the absence of toxin (control; d) and in the presence of
2.5 l M Lqhb1 (s) Appearance of current occurs at more
hyper-polarized potentials in the presence of toxin for oocytes expressing
rNa v 1.2a or rNa v 1.4, but not rNa v 1.5 channels.
Trang 7as LqhIT2 (44–50% identity; Figs 2,3 and 5 [18,20]) The
similarity between Lqhb1 and LqhIT2 is further exemplified
in the effect on the Para NaCh expressed in Xenopus
oocytes The two toxins still differ substantially in that only
Lqhb1 affects mammalian NaChs (Fig 5) These features
may suggest that the group of toxins represented by Lqhb1
and AahIT4 evolved into the anti-insect selective depressant
toxins in the Old World, and into b-toxins presently found
in New World scorpions, after the separation of the
continents It seems that diversification of the b-toxins in
the New World proceeded toward those with affinity for
mammals (e.g Css2 and Css4 [1,12,36]), crustaceans (e.g
Cn5, Cn11, and Cll1 [9–12]) and a group that acquired
a-like activity while maintaining the structural features of
b-toxins (CsEv1–3 [2,12,14])
Tst1, and Tb1 are highly active on mammals and insects
[16], and thus seem to preserve ancient properties of Lqhb1
in the New World
Although all known long chain scorpion toxins share a
similar structural core (a-helix packed against three
anti-parallel b-strands) [2,12,41], and genomic organization
[41,42], the identity of the ancestor polypeptide, from which
they had diverged is a riddle, which is further accentuated
due to difficulties to establish reliable phylogenetic relations
between the available toxin sequences (M Gurevitz &
D Gordon, unpublished observations)
recent description of a long chain polypeptide with only
three disulfide bonds, birtoxin, found in the venom of the
South African scorpion Parabuthus transvaalicus [43,44],
together with the features of Lqhb1 may enable speculation
on a putative route for toxin diversification Birtoxin is toxic
to mice, inhibits Na+currents in dissociated fish retinal cells
in a manner resembling the effect of b-toxins, and shows
substantial sequence similarity to b-toxins from various
New World Centruroides species [43,44] These features
may suggest that birtoxin represents an ancient group of
toxins that could have evolved into the excitatory anti-insect
selective toxins by acquiring a structurally distinctive forth
disulfide bond (Fig 2), or, alternatively, into the group
represented by Lqhb1 by acquiring the forth conserved
disulfide bond found in all but the excitatory toxins The
Lqhb1 group may have evolved after the separation of the
continents to depressant toxins in the Old World, and to
b-toxins in the New World Since the forth disulfide bond
in a and most b-toxins is spatially conserved [2,12,41,45], it
may be hypothesized that toxins with three disulfide bonds,
such as birtoxin, were the progenitors of a-toxins as well
Acknowledgements
This research was supported in part by the United States–Israel
Binational Agricultural Research and Development grant IS-3259–01
(D G and M G.); by the Israeli Science Foundation, grants 508/00
(D G.) and 733/01 (M G.); and by an EU grant QLK3-CT-2000–
00204 (D G and M G.).
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