Characterization of the binding of 125I-labelled d-ACTX-Hv1a to various sodium channels reveals a decrease in affinity for depolarized 0 mV; Kd¼ 6.5 ±1.4 nM vs.polar-ized 55 mV; Kd¼ 0.6
Trang 1Variations in receptor site-3 on rat brain and insect sodium channels highlighted by binding of a funnel-web spider d-atracotoxin
Nicolas Gilles1, Greg Harrison1, Izhar Karbat2, Michael Gurevitz2, Graham M Nicholson3,*
and Dalia Gordon2
1
CEA, De`partement d’Inge`nierie et d’Etudes des Prote`ines, Gif-sur-Yvette, France;2Department of Plant Sciences, Tel Aviv University, Israel;3Department of Health Sciences, University of Technology, Sydney, Australia
d-Atracotoxins (d-ACTXs) from Australian funnel-web
spiders differ structurally from scorpion a-toxins (ScaTx)
but similarly slow sodium current inactivation and compete
for their binding to sodium channels at receptor site-3
Characterization of the binding of 125I-labelled
d-ACTX-Hv1a to various sodium channels reveals a decrease in
affinity for depolarized (0 mV; Kd¼ 6.5 ±1.4 nM)
vs.polar-ized ()55 mV; Kd¼ 0.6 ± 0.2 nM) rat brain synaptosomes
The increased Kdunder depolarized conditions correlates
with a 4.3-fold reduction in the association rate and a
1.8-increase in the dissociation rate In comparison, ScaTx
binding affinity decreased 33-fold under depolarized
condi-tions due to a 48-fold reduction in the association rate The
binding of125I-labelled d-ACTX-Hv1a to rat brain
syna-ptosomes is inhibited competitively by classical ScaTxs and
allosterically by brevetoxin-1, similar to ScaTx binding
However, in contrast with classical ScaTxs,125I-labelled
d-ACTX-Hv1a binds with high affinity to cockroach Na+
channels (Kd¼ 0.42 ± 0.1 nM) and is displaced by the ScaTx, LqhaIT, a well-defined ligand of insect sodium channel receptor site-3 However, d-ACTX-Hv1a exhibits a surprisingly low binding affinity to locust sodium channels Thus, unlike ScaTxs, which are capable of differentiating between mammalian and insect sodium channels, d-ACTXs differentiate between various insect sodium channels but bind with similar high affinity to rat brain and cockroach channels Structural comparison of d-ACTX-Hv1a to ScaTxs suggests a similar putative bioactive surface but a ÔslimmerÕ overall shape of the spider toxin A slimmer shape may ease the interaction with the cockroach and mammalian receptor site-3 and facilitate its association with different conformations of the rat brain receptor, correlated with closed/open and slow-inactivated channel states
Keywords: brevetoxin; sodium channel; spider toxin; syna-ptosomes; voltage-dependent binding
Australian funnel-web spiders (Araneae: Hexathelidae:
Atracinae) are Mygalomorph spiders confined to the
south-eastern seaboard of Australia A number of
neuro-toxins, named atracotoxins (ACTXs) that display various
pharmacological properties, have been isolated from the venom of the funnel-web spider subfamily, Atracinae [1–4] Several lethal atracotoxins that modulate sodium channel gating have been assigned to the d-ACTX group because of their ability to induce spontaneous repetitive firing in neuronal cells, accompanied by plateau action potentials [5–8] The d-ACTXs, d-ACTX-Hv1a (formerly versutoxin [9]), the vertebrate-selective toxin d-ACTX-Hv1b [8] from the venom of Hadronyche versuta, and d-ACTX-Ar1 (formerly robustoxin [10]) from the venom of the male Sydney funnel-web spider Atrax robustus, are highly homologous 42-residue polypeptides These toxins contain
a high proportion of basic residues and show no significant sequence homology with any presently known neurotoxin They are tightly folded molecules constrained by four conserved intramolecular disulfide bonds, arranged in a unique formation The solution structures of d-ACTX-Hv1a and -Ar1 have been determined by NMR spectro-scopy [11,12] and constitute a small triple-stranded antiparallel b-sheet and a Ôcystine knotÕ motive [13] d-ACTXs slow tetrodotoxin (TTX)-sensitive sodium chan-nel inactivation and produce modest shifts in the voltage-dependence of sodium channel activation in insect and mammalian neurons [5–8] in a manner similar to scorpion a-toxins and sea anemone toxins [14,15] Despite the similar effect on sodium current inactivation kinetics, d-ACTXs have a distinct three-dimensional structure, which differs greatly from those of other toxins interacting with
recep-Correspondence to D Gordon, Department of Plant Sciences,
Tel-Aviv University, Ramat-Aviv, Tel Aviv 69978, Israel.
Fax: +972 3 640 6100, E-mail: dgordon@post.tau.ac.il
Abbreviations: Aah-II, antimammalian a-toxin II from the venom of
the scorpion Androctonus australis hector; d-ACTX-Ar1,
d-atraco-toxin-Ar1 (formerly robustoxin) from Atrax robustus; d-ACTX-Hv1a,
d-atracotoxin-Hv1a (formerly versutoxin) from Hadronyche versuta;
ATX-II, toxin II from the sea anemone Anemonia sulcata;
[ 3 H]BTX,[ 3 H]batrachotoxinin, A-20a-benzoate; IC 50 , median
inhibi-tory concentration; K d , dissociation constant; K i , inhibitory constant;
k off , dissociation rate constant; k on , association rate constant; Lqh-II,
Lqh-III, LqhaIT, a classical a-toxin, an a-like toxin, and an a-toxin
highly active on insects, respectively, from the venom of the scorpion
Leiurus quinquestriatus hebraeus; PbTx-1, brevetoxin-1 from the
dinoflagellate Ptychodiscus brevis.
Enzyme: lactoperoxidase (EC 1.11.1.7).
*Present address: Department of Health Sciences, University of
Technology, Sydney PO Box 123, Broadway NSW 2007, Australia.
Fax: +61 2 9514 2228, E-mail: Graham.Nicholson@uts.edu.au
(Received 25 September 2001, revised 15 January 2002, accepted
21 January 2002)
Trang 2tor site-3 {e.g scorpion a-toxins, Aah-II (toxin II from
Androctonus australis hector [16]), LqhaIT (from Leiurus
quinquestriatus hebraeus[17]), and the sea anemone toxin,
anthopleurin-B [18]}
At least seven neurotoxin receptor sites have been
identified on the voltage-gated sodium channel by
radio-labelled toxin binding studies [19] Scorpion a- and sea
anemone toxins such as ATX-II bind to neurotoxin
receptor site-3 (for reviews see [19–22]) The binding of
classical scorpion a-toxins, such as Aah-II [23] and Lqh-II
[24,25], to receptor site-3 on rat brain sodium channels is
voltage dependent and allosterically modulated by
lipid-soluble sodium channel activators such as brevetoxin,
veratridine and batrachotoxin [21,22,27–30] Notably, at
nanomolar concentrations d-ACTX-Ar1 and -Hv1a
com-pletely inhibit the binding of classical scorpion a-toxins
(e.g Aah-II and Lqh-II) to rat brain synaptosomes as well
as the binding of LqhaIT to insect sodium channels
[29,30] Thus, d-ACTXs constitute a unique group of
polypeptides capable of high affinity binding presumably
to receptor site-3 on both mammalian and insect
voltage-gated sodium channels Indeed, they enhance 3
H-batra-chotoxin binding similarly to scorpion a-toxins; however,
they differ from scorpion toxins in that they inhibit, rather
than enhance, the activation of sodium channels by
batrachotoxin [30] Thus, clarification of d-ACTXs
recep-tor sites on sodium channels requires a detailed analysis of
their binding properties
Here we provide a detailed characterization of
radio-labelled d-ACTX-Hv1a direct binding to rat brain and
insect sodium channels We present evidence that
d-ACTX-Hv1a acts similarly to scorpion a-toxins in terms
of its interaction with sodium channels at nanomolar
affinities, similar voltage dependence and allosteric
inter-action with brevetoxin-1 on rat brain sodium channels
Nevertheless, d-ACTX-Hv1a differs from scorpion
a-toxins in its lower voltage dependency and ability to
differentiate between receptor site-3 of cockroach and
locust sodium channels rather than between rat brain and
cockroach
E X P E R I M E N T A L P R O C E D U R E S
Materials
The scorpion a-toxin, Lqh-II, from the venom of the
scorpion L q hebraeus, was purchased from Latoxan (A.P
1724, 05150 Rosans, France) and, in part, was also a
generous gift from Dr P Sautie¨re (Insitut Pasteur, Lille,
France) [24] LqhaIT, an a-insect toxin from the scorpion
L q hebraeus, was produced in Escherichia coli as described
previously [31] Lactoperoxidase (EC 1.11.1.7) was
pur-chased from Sigma Carrier-free Na125I was purchased from
Amersham (Buckinghamshire, UK) All other chemicals
were of analytical grade Filters for binding assays were GF/
C glass fibre discs (Whatman) preincubated in 0.3%
polyethylenimine (Sigma)
Purification of d-ACTX-Hv1a and d-ACTX-Ar1
Crude venom was ÔmilkedÕ by direct aspiration from the
chelicerae of live spiders maintained in a colony, using
silanized (Coatasil; Ajax Chemicals, Australia) glass
pipettes d-ACTX-Hv1a was obtained from adult male or female H versuta spiders while d-ACTX-Ar1 was obtained from adult male A robustus spiders Crude venom was washed from pipettes with 0.1% (v/v) trifluoroacetic acid and d-ACTX-Hv1a and d-ACTX-Ar1 isolated and purified
by RP-HPLC Purification was achieved using a Pharmacia HPLC system using a Vydac analytical rpHPLC column (C18, 250· 4.6 mm, 300 A˚, 5 lm particle size) Pooled venom was applied to the column and venom components eluted at a flow rate of 1 mLÆmin)1using a linear gradient of 5–25% acetonitrile/0.1% trifluoroacetic acid over 22 min, followed by a gradient of 25–50% acetonitrile/0.1% trifluoroacetic acid over 48 min Fractions containing d-ACTX-Hv1a or d-ACTX-Ar1 were then purified further using a linear gradient of 23–32% acetonitrile/0.1% trifluoroacetic acid over 20 min at a flow rate of 1Æml min)1 Toxin quantification was performed using a bicinchoninic acid Protein Assay Kit (Pierce) using BSA as a standard Absorbance was read at 570 nm on a BIO-RAD Model 450 microplate reader The molecular mass was determined by electrospray ionization MS The fractions containing d-ACTX-Hv1a (Mr¼ 4852) or d-ACTX-Ar1 (Mr ¼ 4854) were stored lyophilized at )20 °C in 5–10-nmol aliquots When required, spider toxins were dissolved in 10 mMHepes (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid)/Tris buffer (pH 6.0) and an aliquot
of this stock solution was diluted in the binding solution Any unused d-ACTX stock solution was kept at 4°C and used within 2 weeks
Neuronal membrane preparation All buffers used for preparation of neuronal membranes contained a cocktail of proteinase inhibitors composed of: phenylmethylsulphonyl fluoride (50 lgÆmL)1), pepstatin A (1 lM), iodoacetamide (1 mM) and 1,10-phenantroline (1 mM) All membrane preparation steps were performed
on ice Rat brain synaptosomes were prepared from adult albino Sprague-Dawley rats ( 300 g, laboratory bred), according to the method described by Kanner [32] The synaptosomes, which were concentrated at the 12 and 16% Ficoll gradient interface, were washed and aliquoted into Eppendorf tubes and stored at)80 °C Before experiments, the synaptosomes were rapidly defrosted for 30 s in a 37°C water bath, placed on ice and used immediately (for polarized synaptosomes) For experiments carried out under depolarized conditions, synaptosomes were defrosted and incubated at 37°C for 30 min to facilitate ion gradient dissipation, and then kept on ice until used Insect synaptosomes were prepared from entire heads of adult cockroaches Periplaneta americana according to a pre-viously described method [33] Locust synaptosomes were prepared from dissected brains and ventral nerve cords of adult locusts, Locusta migratoria, as described previously [34,35] Frozen synaptosomes were used within 6 months Membrane protein concentration was determined by a Bio-Rad Protein Assay Kit, using BSA as a standard
Radioiodination of d-ACTX-Hv1, LqhaIT and Lqh-II Toxins (5 lg) were radioiodinated for 1 min using 0.7 IU of lactoperoxidase (EC 1.11.1.7) from bovine milk and 0.5 mCi carrier-free Na125I, in 10 lL HO (diluted 1 : 50 000) and
Trang 350 lL 20 mM phosphate buffer pH 7.2 The
mono-iodo-toxins were purified using a Vydac analytical C18rpHPLC
column and an acetonitrile gradient from 45 to 60% B
(A¼ aqueous 0.1% trifluoroacetic acid, B ¼ 0.085%
tri-fluoroacetic acid, 50% acetonitrile, 0.2% B per min) at a
flow rate of 1 mlÆmin)1 The peak of the mono-iodo LqhaIT
toxin eluted just after the peak of unmodified toxin as
described previously [36] The unmodified d-ACTX-Hv1a,
which eluted at 26% acetonitrile, was followed by two
radiolabelled fractions, eluting at 26.5 and 27.1%
acetonit-rile As d-ACTX-Hv1a contains only two Tyr residues
(positions 22 and 25), we determined the iodinated residue of
each mono-iodo125I-labelled d-ACTX-Hv1a fraction, using
approximately 150 000 c.p.m of each fraction in the
presence of unlabelled toxin by Edman degradation and
sequencing (Applied Biosystem, 477 A protein sequencing),
as described in detail for 125I-labelled LqhaIT [36] The
N-terminal sequence analysis indicated that the first fraction
of125I-labelled d-ACTX-Hv1a was labelled on tyrosine 22,
while the second mono-iodo 125I-labelled d-ACTX-Hv1a
was labelled on tyrosine 25 The concentration of the
radio-labelled toxins were determined according to the specific
activity of125I corresponding to 3000–2500 d.p.m.Æfmol)1
mono-iodotoxin, depending on the age of the radiotoxin
and by estimation of its biological activity as described
previously ([36], usually 60–70% for 125I-labelled LqhaIT
and 35–55% for125I-labelled d-ACTX-Hv1a).125I-labelled
LqhaIT was used within 2 weeks whereas 125I-labelled
d-ACTX-Hv1a was used within 7 days
Competition binding studies
For competition binding experiments using 125I-labelled
d-ACTX-Hv1a on rat brain sodium channels,
synapto-somes were thawed at 37°C (for 30 s) and suspended in
0.2 or 0.6 mL binding buffer containing a low
concentra-tion of radiolabelled toxins (see figure legends) Standard
binding medium composition was (in mM): choline Cl, 130;
CaCl2, 1.8; KCl, 5; MgSO4, 0.8; Hepes, 50;D-glucose 10;
and 2 mgÆmL)1 BSA Following incubation for the
designated time periods the reaction was terminated by
dilution with 2 ml ice-cold wash buffer of the following
composition (in mM): choline Cl, 140; CaCl2, 1.8; KCl, 5.4;
MgSO4, 0.8; Hepes, 50; pH 7.2 and 5 mg mL)1 BSA
Separation of free from bound toxin was achieved by rapid
filtration under vacuum using Whatman GF/C filters
preincubated with 0.3% polyethylenimine The filter discs
were then rapidly washed twice with 2 mL buffer
Termination of the reaction and washing lasted 10 s
Nonspecific toxin binding was determined in the presence
of a high concentration of the unlabelled toxin, as specified
in figure legends, and comprised typically 5–10% of total
binding for 125I-labelled LqhaIT and 30–50% for 125
I-labelled d-ACTX-Hv1a Competition binding experiments
using125I-labelled LqhaIT and125I-labelled d-ACTX-Hv1a
on insect neuronal membranes were performed according
to established methods [35,36] under conditions specified in
the figure legends
Equilibrium and kinetic analysis of binding
The median inhibitory concentration (IC50) values for the
inhibition of toxin binding were determined by nonlinear
regression analysis using the Hill equation using a Hill coefficient (nH) of 1 Mathematical curve fitting was accomplished using KALEIDAGRAPH (Synergy Software, USA) for IC50 determination and the Ki values were calculated [37] Cold saturation assays were performed using increasing concentrations of the unlabelled toxin in the presence of a constant low concentration of the mono-iodinated toxin Hot saturation assays were performed using increasing concentrations of the radiolabelled toxin, with the same amount of membranes Data were analysed using the iterative programLIGAND(Elsevier Biosoft) using Ôcold saturationÕ or Ôhot saturationÕ analysis The kinetic data for ligand association and dissociation rates were subjected to analysis by LIGAND, using Ôkinetic analysisÕ Each curve was subjected to multislope analysis to detect the presence of one or two slopes Toxin dissociation curves were initiated by the addition of excess unlabelled toxin and the dissociation rate constant (koff) was determined directly from a first order plot of ligand dissociation vs time The rate of toxin association (kon) was determined from the equation:
kon ¼ kobs
½RLe
½L½RLmax
where [L] is the concentration of ligand, [RL]e is the concentration of the complex at equilibrium, [RL]maxis the maximum number of receptors present (determined in a parallel saturation experiment) and kobsis the slope of the pseudo-first order plot of ln ([RL]e/{[RL]e–[RL]t}) vs time [38] The concentration of labelled ligand in association kinetic determinations was adjusted to keep the reaction at pseudo-first order conditions and varied according to the Kd values of the toxin under polarized or depolarized condi-tions [38] Results were compared using a Student’s t-test and all data are expressed as the mean ± SEM from the number of experiments (n) indicated The corresponding affinity (Kd) can be calculate from the kinetics parameters according to the equation Kd¼ koff/kon
R E S U L T S
Both d-ACTX-Hv1a and d-ACTX-Ar1 inhibit completely the binding of 125I-labelled Aah-II [28] and 125I-labelled Lqh-II [29] to rat brain sodium channels In order to characterize the receptor binding site for d-ACTXs on sodium channels, we analysed the binding properties of radioiodinated d-ACTX-Hv1a to rat brain synaptosomes
We have first examined the ability of the classical scorpion a-toxin, Lqh-II, to displace 125I-labelled d-ACTX-Hv1a from its binding site and found a Ki value of 0.25 ± 0.03 nM (n¼ 4; data not shown), supporting the notion that d-ACTXs share receptor site-3 with scorpion a-toxins
Evidence that d-ACTX-Hv1a binding to rat brain synaptosomes is voltage dependent
Binding of scorpion a-toxins depends on polarization of the synaptosome membrane and therefore is a useful measure in monitoring membrane potential Indeed, a 90% decrease of the initial maximal binding between polarized and depolarized synaptosomes has been shown [39–42]
Trang 4The resting membrane potential of rat brain synaptosomes
is approximately)55 mV (at 5 mM[K+]o) due mainly to a
high intracellular concentration of K+, which diffuses
passively through the membrane [39,41,43] Although
depolarization of synaptosomes by elevating [K+]0is often
used for measuring the influence of membrane potential on
scorpion a-toxin binding [40,44], we have found that high
concentrations of K+ in the binding buffer perturb the
binding of125I-labelled d-ACTX-Hv1a (data not shown)
Unlike the situation with the binding of the a-toxin,
125I-labelled Lqh-II, where nonspecific binding did not
change with increasing external K+ concentration
(bet-ween 5 and 135 mM[42]), the level of nonspecific binding
of 125I-labelled d-ACTX-Hv1a varied greatly, posing
difficulties for data analysis Therefore, in order to
depolarize the membrane without affecting other binding
conditions, the synaptosomes were incubated for 30 min at
37°C in normal binding buffer (containing 5 mMK+see
Experimental procedures) prior to addition of the labelled
toxin (under such conditions the K+ gradient dissipates,
the membrane potential approaches 0 mV and scorpion
a-toxin binding is decreased by 90% [42]) The time-course
of 125I-labelled d-ACTX-Hv1a binding to polarized rat
brain synaptosomes was performed at 22°C to maintain
the resting membrane potential for longer duration
(Fig 1A [42]) Maximal binding was achieved after
10–15 min and was maintained for an additional 10 min
before an apparent decrease could be observed A similar
decrease in saturable binding was observed with the
scorpion a-toxin Lqh-II (Fig 1, inset), suggesting
depend-ency of binding on membrane depolarization for both
toxins Therefore, all subsequent experiments on polarized synaptosomes were performed at 22°C with a 20 min incubation time to reach equilibrium binding conditions Despite the similar effect of membrane potential on binding of both toxins, the ratio between 125I-labelled d-ACTX-Hv1a (Fig 1B, left bars) maximal binding to polarized (empty bar) vs depolarized (gray bars) synapto-somes was substantially different from that measured for
125I-labelled Lqh-II (Fig 1B, right bars) This difference necessitated analysis of the binding affinity d-ACTX-Hv1a
to rat brain synaptosomes under polarized and depolarized conditions
Affinity of d-ACTX-Hv1a for polarized and depolarized rat brain synaptosomes
To study the influence of synaptosome membrane potential
on 125I-labelled d-ACTX-Hv1a affinity, 125I-labelled d-ACTX-Hv1a was incubated with polarized or depolarized rat brain synaptosomes in the presence of increasing concentrations of unlabelled toxin (cold saturation) The dissociation constant (Kd) of d-ACTX-Hv1a increased 11-fold between polarized (Kd¼ 0.57 ± 0.20 nM; n ¼ 3) and depolarized (Kd¼ 6.5 ± 1.4 nM; n¼ 5) synapto-somes, whereas the maximum number of receptor sites (Bmax) increased 1.8-fold (P < 0.05; Bmax¼ 1.24 ± 0.17 pmolÆmg protein)1; n¼ 3 and 2.26 ± 0.05 pmolÆmg protein)1; n¼ 5, respectively; Fig 2) To assure the signi-ficance of the change in Bmax, the experiments under polarized and depolarized conditions were performed in parallel using the same batch of rat brain synaptosomes It is
Fig 1 Time-course of125I-labelled d-ACTX-Hv1a and125I-labelled Lqh-II binding to rat brain synaptosomes at 22 °C (A) Typical association kinetics of 125 I-labelled d-ACTX-Hv1a (75 p M ) to polarized synaptosomes (20 lgÆmL)1membrane protein) Non-specific binding, determined in the presence of 1 l M Lqh-II, was time-invariant, and was subtracted from the experimental data points Maximal binding of125I-labelled d-ACTX-Hv1a remained stable for 10 min before decreasing due to spontaneous depolarization of synaptosomes The time-course of 60 p M125I-labelled Lqh-II binding to polarized synaptosomes (20 lgÆmL)1) is presented in the inset as per cent of maximal specific binding (B) Comparison of the maximal binding of125I-labelled d-ACTX-Hv1a (left bars) and125I-labelled Lqh-II (right bars) to polarized (empty bars) and to depolarized (gray bars) synaptosomes 125 I-labelled d-ACTX-Hv1a binding was performed as described for panel (A) Maximal binding under polarized (5.5 ± 0.3 p M and 5.1 ± 0.2 p M bound 125 I-labelled d-ACTX-Hv1a or 125 I-labelled Lqh-II, respectively) and depolarized synaptosomes pre-treated at 37 °C for 30 min (3.8 ± 0.6 p M and 0.75 ± 0.15 p M , for125I-labelled d-ACTX-Hv1a or125I-labelled Lqh-II, respectively), corresponds
to the level of125I-labelled toxin binding after 20 and 60 min of incubation for polarized and depolarized conditions, respectively.
Trang 5noteworthy, that in order to maintain a pseudo-first order
reaction conditions, toxin and receptor (membrane protein)
concentrations were adjusted as a function of the change in
125I-labelled d-ACTX-Hv1a affinity (Fig 2 [38])
Kinetic constants of d-ACTX-Hv1a binding to rat brain
synaptosomes
125I-Labelled d-ACTX-Hv1a was incubated with
synapto-somes and the association binding kinetics were monitored
until equilibrium had been reached (Fig 3A, closed symbols
for polarized, and open symbols for depolarized
synapto-somes, respectively) After 10 min incubation with polarized
synaptosomes, toxin dissociation was initiated by adding
1 lMunlabelled d-ACTX-Hv1a (Fig 3B, closed symbols)
The calculated association and dissociation rate constants,
k and k , under polarized membrane conditions were
1.84 ± 0.2· 106ÆM )1Æs)1(n¼ 3) and 1.1 ± 0.1 · 10)3Æs)1 (n¼ 3), respectively The corresponding Kd(0.6 ± 0.1 nM) calculated from the kinetic values, was comparable with the values obtained at equilibrium (Fig 2) Equilibrium of d-ACTX-Hv1a binding to depolarized synaptosomes was achieved after longer incubation (Fig 3A, open symbols), and dissociation was induced after 60 min of association by adding 1 lM unlabelled d-ACTX-Hv1a (Fig 3B, open symbols) The calculated kon and koff under depolarized membrane conditions were 0.43 ± 0.13· 106ÆM )1Æs)1 (n¼ 3) and 2.0 ± 0.3 · 10)3s)1(n¼ 3), respectively The corresponding calculated Kd was 4.7 ± 2.1 nM, which fitted the value obtained at equilibrium These results indicate that d-ACTX-Hv1a binding is dependent on the membrane potential of synaptosomes Interestingly, syna-ptosome depolarization had a minute effect on koff but decreased fourfold the kon These results support our recent studies using the classical scorpion a-toxin, Lqh-II [42], and seem to provide a different interpretation to that suggested previously for scorpion a-toxins, which attributed the change in binding affinity under depolarized conditions mainly to an increase in the dissociation rate [15,26,39,40,44–47]
Allosteric modulation of d-ACTX-Hv1a binding site
on rat brain synaptosomes Brevetoxin-1 (PbTx-1) from a marine dinoflagellate, inhibits allosterically the binding of the scorpion a-toxin, Aah-II, to rat brain synaptosomes [27,28] To examine the similarity in binding to receptor site-3 between scorpion a-toxins and d-ACTXs, we analysed the effect of PbTx-1 on125I-labelled d-ACTX-Hv1a binding to rat brain synaptosomes Simi-larly to the effect of PbTx-1 on Aah-II binding [27,28], this brevetoxin substantially inhibited the binding of
125I-labelled d-ACTX-Hv1a with an IC50 of 50 nM (data not shown) We also analysed the effect of deltamethrin, a pyrethroid insecticide known to modulate sodium channels, and like scorpion a-toxins [48] found that it had no allosteric effect on d-ACTX-Hv1a binding
d-ACTX-Hv1a differentiates between cockroach and locust sodium channels
d-Atracotoxins are unique in their potency to displace scorpion a-toxins from their binding sites on both rat brain and cockroach sodium channels [29,30] Therefore, the interaction of 125I-labelled d-ACTX-Hv1a with scorpion a-toxins on binding to cockroach neuronal membranes was examined Competition binding experiments using increas-ing concentrations of the scorpion toxins, Lqh-II, LqhaIT, and Lqh-III as well as the related spider toxin, d-ACTX-Ar1, revealed complete inhibition of125I-labelled d-ACTX-Hv1a binding by all toxins tested (Fig 4, main panel) Scatchard transformation of the competition binding curve of d-ACTX-Hv1a to cockroach neuronal membranes (cold saturation, Fig 4, inset) provided a Kd value of 0.42 ± 0.1 nM (n¼ 3), which was highly similar to the affinity of d-ACTX-Hv1a binding to polarized rat brain synaptosomes (Table 1) The receptor site capacity (Bmax¼ 2.1 ± 0.5 pmolÆmg protein)1; n¼ 3) was similar to that obtained previously for LqhaIT binding to cockroach neuronal membranes [35,36]
Fig 2 Scatchard plots of 125 I-labelled d-ACTX-Hv1a binding to rat
brain synaptosomes (A) 92 p M125I-labelled d-ACTX-Hv1a incubated
at 22 °C for 20 min with polarized synaptosomes (28.8 lgÆmL)1) and
(B) 168 p M125I-labelled d-ACTX-Hv1a incubated at 22 °C for 60 min
with depolarized synaptosomes (36.5 lgÆmL)1), in the presence of
increasing concentrations of unlabelled toxin (cold saturation) (see
Experimental procedures) Analysis of a typical experiment is
presen-ted Nonspecific binding, determined in the presence of 0.2 l M (A) or
1 l M (B) d-ACTX-Hv1a, was subtracted Equilibrium binding
parameters were calculated using the program LIGAND (see
Experi-mental procedures) The dissociation constants (K d ) were
0.57 ± 0.2 n M and 6.5 ± 1.4 n M and the maximum number of
binding sites (B max ) were 1.24 ± 0.17 pmolÆmg protein)1 and
2.26 ± 0.05 pmolÆmg protein)1, under polarized (n ¼ 3) and
depo-larized (n ¼ 5) conditions, respectively.
Trang 6Unexpectedly, however, no specific binding of
125I-labelled d-ACTX-Hv1a to locust neuronal membranes
could be detected As we have recently demonstrated that
iodination of one Tyr residue in the a-like toxin, Lqh-III, impairs binding to locust but not cockroach sodium channels [36], we identified which Tyr residue was iodinated on 125I-labelled d-ACTX-Hv1a Amino acid sequence analysis of the two radiolabelled peaks obtained during toxin radioiodination (see Experimental proce-dures) identified an iodinated Tyr22 in the first peak and an iodinated Tyr25 in the second peak Both iodinated derivatives did not differ in their binding properties to cockroach neuronal membranes (data not shown) In order
to eliminate the possibility that the lack of 125I-labelled d-ACTX-Hv1a binding to locust neuronal membranes was consequent on its iodination per se, we examined the binding of d-ACTX-Hv1a to locust sodium channels indirectly, by its ability to compete for 125I-labelled LqhaIT binding (Fig 5) Interestingly d-ACTX-Hv1a competed for LqhaIT binding only at high concentrations (Ki¼ 67 ± 17 nM; n¼ 3) with a Kivalue 160-fold higher than the Kdfor cockroach sodium channels (Fig 4) Thus,
in contrast with the scorpion a-like toxin Lqh-III [36] and despite its high binding affinity for cockroach sodium channels, d-ACTX-Hv1a is a weak ligand on locust sodium channels (Fig 5)
D I S C U S S I O N
Effect of membrane depolarization on kinetics
of toxin binding to receptor site-3 The binding properties of the spider toxin, d-ACTX-Hv1a,
to rat brain synaptosomes resemble those of scorpion a-toxins, thereby suggesting a common receptor binding site
on the sodium channel This resemblance is substantiated by
a similar, yet nonidentical, decrease in binding affinity at polarized ()55 mV) and depolarized (0 mV) membrane potentials (Kdincrease of 11.4-fold for d-ACTX-Hv1a and 33-fold for the classical a-toxin, Lqh-II, Table 1 [42,49]) The increase in Kdof d-ACTX-Hv1a binding correlates with
a 4.3-fold lower association rate and a 1.8-fold increase in the dissociation rate (Table 1) The more profound increase
Fig 3 Association (A) and dissociation (B) kinetics of125I-labelled d-ACTX-Hv1a binding to polarized and depolarized rat brain synaptosomes Fifty and 200 p M125I-labelled d-ACTX-Hv1a were incubated at 22 °C (in 200 lL) in the presence of 37 or 73 lgÆmL)1polarized or depolarized synaptosomes, respectively, for various periods of time Nonspecific binding, determined in the presence of 200 n M or 1 l M Lqh-II (for polarized and depolarized conditions, respectively) was time-invariant and was subtracted from the experimental points.125I-labelled d-ACTX-Hv1a dissociation was initiated by addition of 200 n M or 1 l M unlabelled toxin after 10 or 60 min association under polarized and depolarized conditions, respectively (B) A typical experiment is presented Kinetic constants, representing the mean of three experiments, were: k on ¼ 1.84 ± 0.2 · 10 6
s–1Æ M )1 and k off ¼ 1.1 ± 0.1 · 10 –3
s)1 under polarized conditions; k on ¼ 0.43 ± 0.13 · 10 6
s)1Æ M )1 and k off ¼ 2.0 ± 0.3 · 10 –3 s)1under depolarized conditions.
Fig 4 Binding interaction of125I-labelled d-ACTX-Hv1a with
cock-roach sodium channels Competition for 125 I-labelled d-ACTX-Hv1a
(120 p M ) binding to neuronal membranes (7 lgÆmL)1) by various
neurotoxins Nonspecific binding, determined in the presence of
200 n M LqhaIT, was subtracted Bound 125 I-labelled d-ACTX-Hv1a is
expressed as the percentage of maximal specific binding in the absence
of competitor toxins The competition curves were fitted by the
non-linear Hill equation (with a Hill coefficient of 1) to determine IC 50
values (see Experimental procedures) Typical curves are presented.
The K i values (in n M ) and the number of experiments (n) are: LqhaIT,
0.12–0.16 (n ¼ 2); Lqh-III, 0.12–0.14 (n ¼ 2); d-ACTX-Hv1a,
2.6–3.0 (n ¼ 2); d-ACTX-Ar1, 1.5–2.5 (n ¼ 2); Lqh-II, 9.5–14.1 n M
(n ¼ 2) Inset: Scatchard transformation of 55 pM 125
I-labelled d-ACTX-Hv1a binding to cockroach neuronal membranes
(8.7 lgÆmL)1) in a volume of 600 lL using increasing concentrations
of unlabelled d-ACTX-Hv1a (Ôcold saturationÕ) The equilibrium
binding parameters were calculated using the program LIGAND Data
represents the mean of two cold- and two hot-saturation experiments
(see Experimental procedures),which showed no significant differences.
K ¼ 0.42 ± 0.1 n ; B ¼ 2.1 ± 0.5 pmolÆmg protein)1.
Trang 7in Kdof Lqh-II binding under similar steady-state
condi-tions may be related to the 48-fold decrease in its association
rate constant (Table 1) Thus, the conformational alteration
induced by depolarization at receptor site-3 appears to
affect toxin binding by two mechanisms The first involves
steric (architectural) and/or electrostatic (long-range)
chan-ges, which are unfavourable for d-ACTX-Hv1a access and
even more so for Lqh-II, thus reducing substantially the kon
The difference in konsuggests that the two toxins bind in a
nonidentical manner to overlapping receptor sites The
second mechanism involves a change in the surface of
receptor site-3, which destabilizes its close fit with the bound
toxin, thus increasing the off-rate Surprisingly, this change
is much smaller as the off-rate of both toxins increased less
then twofold between polarized and depolarized
steady-state conditions but affects d-ACTX-Hv1a binding affinity
more than that of Lqh-II (Table 1) Hence, depolarization
conditions hinder Lqh-II association to a greater extent than d-ACTX-Hv1a, and may be related to their different structures
A slow inactivated channel state prevails
in depolarized synaptosomes d-ACTX-Hv1a binding to rat brain synaptosomes reveals
an increase in Kd(11.4-fold) and in Bmax(1.8-fold) between polarized and depolarized conditions (Fig 2) The increase
in Bmaxmay be attributed to a change in the ratio between sodium channels at high and low affinity states for toxin binding We assume that at resting membrane potentials, only 60% of site-3 receptors are in a high affinity conformation enabling toxin binding (presumably on sodium channels in closed states [47]), whereas the remain-ing channels are in a low affinity conformation associated with the slow-inactivated state This suggestion is supported
by our study with Lqh-II, using both binding and electro-physiological analyses [42] and the study of Smith & Goldin [50] which implied that, at)55 mV, most rat brain subtype I (rBI) channels, which comprise 20% of sodium channels
in synaptosomes [51], were available for activation presum-ably by being in closed, resting states [50] Nevertheless they showed that at identical membrane potential more than 50% of brain subtype IIA (rBIIA) channels that constitute the majority ( 80%) in synaptosomes [51], were in an inactivated state Our electrophysiological analysis of rBII channels expressed in mammalian cells supports this conclusion [42] Thus, a substantial fraction of sodium channels would occupy the slow-inactivated states at polarized synaptosomes and thus display a low affinity conformation for toxin binding [42] The observed increase
in Aah-II binding to rat brain sodium channels in the presence of TTX [28] may be attributed to shifting receptor site-3 from low to high affinity conformation by binding of TTX to the external vestibule of the slow-inactivated channel pore In so far as the125I-labelled d-ACTX-Hv1a concentrations used in the binding studies were low compared to the Kd(Figs 2 and 3), the low affinity binding sites in polarized synaptosomes were undetectable, because only a small fraction of site-3 receptors were occupied Conversely, in depolarized synaptosomes, most sodium channels are in the slow-inactivated states, thus available for toxin binding only at low affinity conformations of receptor site-3 In this situation and under proper ligand
concentra-Table 1 Comparison between equilibrium and kinetic binding parameters of 125 I-labelled d-ACTX-Hv1a and the scorpion a-toxin, Lqh-II Binding to polarized (membrane potential of )55 mV) and depolarized (0 mV) synaptosomes is performed as described in Figs 2 and 3 (see text for details) Data are means ± SEM values n ¼ number of independent experiments Data for Lqh-II binding parameters are from Gilles et al [42].
K d
(n M )
k on
(106M )1 Æs)1)
k off
(10–3Æs)1)
Fig 5 Competition of d-ACTX-Hv1a for125I-labelled LqhaIT binding
to locust sodium channels.125I-labelled LqhaIT (0.2 n M ) was incubated
for 60 min at 22 °C with increasing concentrations of LqhaIT or
d-ACTX-Hv1a, and the binding to locust neuronal membranes
(60 lgÆmL)1) was determined Nonspecific binding, determined in the
presence of 200 n M LqhaIT, was subtracted Bound 125 I-labelled
LqhaIT is expressed as the percentage of the maximal specific binding
in the absence of competitor A typical experiment is presented The
competition curves were fitted by a nonlinear Hill equation (with a Hill
coefficient of 1) to determine IC 50 values (see Experimental
proce-dures) The calculated K i values [37] were: LqhaIT, 1.2–2.2 n M
(n ¼ 2); d-ACTX-Hv1a, 67 ± 17 n M (n ¼ 3).
Trang 8tions, maximum binding capacity is observed (Fig 2, and
see [42])
The coexistence of (at least) two distinct conformational
states of receptor site-3 among sodium channels in
polarized synaptosomes gains further support from the
1.9-fold increase in receptor site capacity for scorpion
a-toxins binding in the presence of batrachotoxin, an
alkaloid toxin binding to receptor site-2 [41] This result
suggests that batrachotoxin allosterically affects receptor
site-3 by shifting it from the low to the high affinity state,
increasing both scorpion a-toxin affinity and receptor site
capacity [20,21,28] Together these results indicate that the
low affinity conformation of receptor site-3 involves
changes in external channel regions, which are affected
by alterations in membrane potential or binding of toxins
to topologically distinct receptor sites on the channel
protein [28] The mechanisms involved in this affinity
change are different, however, as depolarization affects
mainly the association rate whereas allosteric modulation
by other toxins affects mainly the dissociation rate
constant [21,27,41]
Resemblance of the putative bioactive surfaces
of LqhaIT, Aah-II, and d-ACTX-Hv1a
Despite the difference in three-dimensional structure,
sequence, and size, the similarity in binding properties of
scorpion a-toxins and d-ACTX-Hv1a suggests some
struc-tural resemblance at the bioactive surface However, the
variations in kon and unusual binding selectivity of the
toxins to receptor site-3 may result from either variations at
the bioactive surface, or other, yet unidentified, structural
differences In search for possible resemblance of molecular
exteriors, we compared LqhaIT [17], Aah-II (which is
almost identical to Lqh-II [16,24]) and d-ACTX-Hv1a [1]
focusing on residues shown in LqhaIT to constitute the
bioactive surface (Fig 6, left [52,53]) A number of bioactive
residues of LqhaIT appear also on the surface of Aah-II in a
similar position, and interestingly, also appear on the
surface of d-ACTX-Hv1a (Fig 6) The positively charged
Lys3, Lys4, Arg5, and Lys10 of d-ACTX-Hv1a are oriented
similarly to Lys8, Arg58, Lys62, and Arg18 of LqhaIT The
aromatic Trp7 and Tyr25 in d-ACTX-Hv1a resemble to
some extent Trp38 and Phe17 in LqhaIT or Trp38 and
Phe15 in Aah-II The nonpolar Asn6 in d-ACTX-Hv1a occupies a similar position to Asn44 in LqhaIT or Aah-II Finally, the negatively charged Glu12 in d-ACTX-Hv1a resembles Glu24 in both LqhaIT and Aah-II
Despite this similarity, the shape of d-ACTX-Hv1a at the angle presented in Fig 6, is slimmer than that of the scorpion toxins, which may explain its accessibility, with high binding affinity, to both rat brain and cockroach receptor site-3 This possibility may also explain the smaller decrease in association rate to receptor site-3 between polarized and depolarized rat brain synaptosomes (Fig 2) compared to Lqh-II Conformational changes in the sodium channel states that are associated with a shift from the high
to the low affinity state of receptor site-3 by depolarization may involve steric hindrance for toxin access, which is less pronounced for the slimmer d-ACTX This hypothesis is in concert with the smaller depolarization effect on the association rate, kon(Fig 3) of d-ACTX compared to that
of Lqh-II (Table 1) In light of the structural resemblance at the putative bioactive surface between the spider and scorpion toxins, the subtle variations in action and binding properties [7,30], suggest that d-ACTXs interact with the sodium channel at a nonidentical, yet overlapping site to that of scorpion a-toxins
Differences between cockroach and locust receptor site-3
All of the site-3 toxins that compete for classical scorpion a-toxin binding to rat brain sodium channels compete for LqhaIT binding to cockroach sodium channels but with different potencies [29,30,35] The spider d-ACTXs are unique in that they bind with equally high affinity to receptor site-3 of both rat brain and cockroach sodium channels (Figs 2 and 4 [29,30]) Such broad potency for sodium channels of distant phyla could be related to the slimmer shape of the spider toxin compared with the bulkier appearance of scorpion a-toxins (Fig 6)
d-ACTXs are similar to LqhaIT in toxicity symptoms of injected insects [30,35,54], and the inactivation of the sodium current in cockroach neuronal preparations [5,54] Therefore, the substantial difference in d-ACTX-Hv1a binding affinity for cockroach vs locust sodium channels
is surprising Structural differences between the two insect
Fig 6 Structural comparison of scorpion a-toxins and d-atracotoxin-Hv1a The structures for Aah-II ([16], PDB accession code 1PTX), d-ACTX-Hv1a ([11]; 1VTX); and LqhaIT ([17]; 1LQH) are presented with a similar orientation of their putative bioactive surfaces Residues reported to participate in the bioactive surface of LqhaIT [52,53] together with topologically related residues in Aah-II and d-ACTX-Hv1a are highlighted and colour coded: blue, positively charged; green, aromatic; magenta, Asn; yellow, negatively charged (see text for details) Toxin models were prepared
Trang 9sodium channels have been previously inferred from
allosteric modulations of LqhaIT binding Brevetoxin
(site-5 toxin) and veratridine (site-2 toxin) increase the
binding of LqhaIT to locust but not to cockroach sodium
channels [27,35,55] In addition, we have shown that
Lqh-III, which binds to locust and cockroach receptor site-3
equally well, lost its binding capacity to locust sodium
channels in its iodinated form [36] Regardless of iodination,
however, the spider toxin binds with high affinity to
cockroach and rat brain channels and with low affinity to
locust receptor site-3 Of note is that LqhaIT and Lqh-III
bind very poorly to rat brain sodium channels [49,56] in
contrast with the high affinity binding of d-ACTX-Hv1a
(Table 1) Hence, various toxin probes may expose subtle
differences at receptor site-3 Still, the structural basis for
these selective interactions requires determination of contact
surfaces between the various ligands and their receptor
binding sites
Concluding remarks
These results suggest that toxin selectivity for receptor site-3
may be conferred not only by structural variables at the
direct interacting surface, but also by external channel
elements that may affect toxin access to the binding site We
have shown that toxins with different shapes or electrostatic
surface potentials are capable of reaching the same or
overlapping receptor sites (this study and see also
[35,42,56]) Thus, structural differences in channel regions
that flank the putative binding site could also be involved in
toxin binding This assumption is supported by the
differ-ence between the association rates of Lqh-II and d-ACTX
to polarized synaptosomes (which mainly account for the
difference in their affinity) and the changes detected in konto
different channel states (see Table 1) Also, the differences in
association rates of Lqh-II, Lqh-III and LqhaIT to rat
skeletal muscle channels, expressed in mammalian cells [46],
support this notion The rational design of subtype-specific
compounds will take into account such architectural
considerations, which may facilitate the design of subtype
specific drugs and insecticides
A C K N O W L E D G E M E N T S
This work was supported in part by an Australian Research Council
research grant and an UTS internal research grant (to G M N.), by a
research grant from the Israeli Science Foundation (508/00, to D G.)
and by grants from BARD, The United States-Israel Binational
Agricultural Research & Development (IS-2901–97C, to M G and
IS-3259–01 to D G.).
R E F E R E N C E S
1 Fletcher, J.I., Smith, R., O’Donoghue, S.I., Nilges, M., Connor,
M., Howden, M.E.H., Christie, M.J & King, G.F (1997) The
structure of a novel insecticidal neurotoxin,
omega-atracotoxin-HV1, from the venom of an Australian funnel web spider Nat.
Struct Biol 4, 559–566.
2 Wang, X.-H., Smith, R., Fletcher, J.I., Wilson, H., Wood, C.J.,
Howden, M.E.H & King, G.F (1999) Structure–function studies
of omega-atracotoxin, a potent antagonist of insect voltage-gated
calcium channels Eur J Biochem 264, 488–494.
3 Szeto, T.H., Wang, X.-H., Smith, R., Connor, M., Christie, M.J.,
Nicholson, G.M & King, G.F (2000) Isolation of a funnel web
spider polypeptide with homology to mamba intestinal toxin 1 and the embryonic head inducer Dickkopf1 Toxicon 38, 429–442.
4 Wang, X.-H., Connor, M., Smith, R., Maciejewski, M.W., Howden, M.E.H., Nicholson, G.M., Christie, M.J & King, G.F (2000) Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge Nat Struct Biol 7, 505–513.
5 Grolleau, F., Stankiewicz, M., Birinyi-Strachan, L.C., Wang, X.-H., Nicholson, G.M., Pelhate, M & Lapied, B (2001) Elec-trophysiological analysis of the neurotoxic action of a funnel-web spider toxin, d-atracotoxin-Hv1a, on insect voltage-gated sodium channels J Exp Biol 204, 711–721.
6 Nicholson, G.M., Willow, M., Howden, M.E.H & Narahashi, T (1994) Modification of sodium channel gating and kinetics by versutoxin from the Australian funnel-web spider Hadronyche versuta Pflugers Arch 428, 400–409.
7 Nicholson, G.M., Walsh, R., Little, M.J & Tyler, M.I (1998) Characterisation of the effects of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, on sodium channel activation and inactivation Pflugers Arch 436, 117–126.
8 Szeto, T.H., Birinyi-Strachan, L.C., Wang, X.-H., Smith, R., Connor, M., Christie, M.J., King, G.F & Nicholson, G.M (2000) Isolation and pharmacological characterisation of d-atracotoxin-Hv1b, a vertebrate-selective sodium channel toxin FEBS Lett.
470, 293–299.
9 Brown, M.R., Sheumack, D.D., Tyler, M.I & Howden, M.E.H (1988) Amino acid sequence of versutoxin, a lethal neurotoxin from the venom of the funnel-web spider Atrax versutus Biochem.
J 250, 401–405.
10 Sheumack, D.D., Claassens, R., Whiteley, N.M & Howden, M.E.H (1985) Complete amino acid sequence of a new type of lethal neurotoxin from the venom of the funnel-web spider Atrax robustus FEBS Lett 181, 154–156.
11 Fletcher, J.I., Chapman, B.E., Mackay, J.P., Howden, M.E.H & King, G.F (1997) The structure of versutoxin (d-atracotoxin-Hv1): implications for binding of site-3 toxins to the voltage-gated sodium channel Structure 5, 1525–1535.
12 Pallaghy, P.K., Alewood, D., Alewood, P.F & Norton, R.S (1997) Solution structure of robustoxin, the lethal neurotoxin from the funnelweb spider Atrax robustus FEBS Lett 419, 191–196.
13 Pallaghy, P.K., Neilsen, K.J., Craik, D.J & Norton, R.S (1993) A common structural motif incorporating a cystine knot and a triple-stranded beta-sheet in toxic and inhibitory polypeptides Protein Sci 3, 1833–1839.
14 Hanck, D.A & Sheets, M.F (1995) Modification of inactivation
in cardiac sodium channels: ionic current studies with antho-pleurin-A toxin J Gen Physiol 106, 601–616.
15 Strichartz, G.R & Wang, G.K (1986) Rapid voltage-dependent dissociation of scorpion a-toxins coupled to Na channel inacti-vation in amphibian myelinated nerves J Gen Physiol 88, 413–435.
16 Fontecilla-Camps, J.-L., Habersetzer-Rochat, C & Rochat, H (1988) Orthorhombic crystals and three dimensional structure of the potent toxin II from the scorpion Androctonus Australis Hector Proc Natl Acad Sci USA 85, 7443–7447.
17 Tugarinov, V., Kustanovich, I., Zilberberg, N., Gurevitz, M & Anglister, J (1997) Solution structures of a highly insecticidal recombinant scorpion alpha-toxin and a mutant with increased activity Biochemistry 36, 2414–2424.
18 Monks, S.A., Pallaghy, P.K., Scanlon, M.J & Norton, R.S (1995) Solution structure of the cardiostimulant polypeptide antho-pleurin-B and comparison with anthopleurin-A Structure 3, 791–803.
19 Gordon, D (1997) A new approach to insect-pest control – combination of neurotoxins interacting with voltage sensitive sodium channels to increase selectivity and specificity Invertebr Neurosci 3, 103–116.
Trang 1020 Catterall, W.A (1986) Molecular properties of voltage-sensitive
sodium channels Annu Rev Biochem 55, 953–985.
21 Catterall, W.A (1992) Cellular and molecular biology of
voltage-gated sodium channels Physiol Rev 72 (Suppl.), S15–S48.
22 Gordon, D (1997) Sodium channels as targets for neurotoxins:
mode of action and interaction of neurotoxins with receptor sites
on sodium channels In Toxins and Signal Transduction
(Lazarowici, P & Gutman, Y., eds), pp 119–149 Harwood Press,
Amsterdam, the Netherlands.
23 Martin-Eauclaire, M.-F & Couraud, F (1995) Scorpion
neuro-toxins: Effects and mechanisms In Handk Neurotoxicology
(Chang, L.W & Dyer, R.S., eds), pp 683–716 Marcel Dekker,
New York.
24 Sautie¨re, P., Ceste¨le, S., Kopeyan, C., Martinage, A., Drobecq, H.,
Doljansky, Y & Gordon, D (1998) New toxins acting on sodium
channels from the scorpion Leiurus quinquestriatus hebraeus
suggest a clue to mammalian vs insect selectivity Toxicon 36,
1141–1154.
25 Gordon, D., Savarin, P., Gurevitz, M & Zinn-Justin, S (1998)
Functional anatomy of scorpion toxins affecting sodium channels.
J Toxicol Toxin Rev 17, 131–159.
26 Catterall, W.A (1977) Activation of the action potential Na+
ionophore by neurotoxins J Biol Chem 252, 8669–8676.
27 Ceste¨le, S., Ben Khalifa, R., Pelhate, M., Rochat, H & Gordon,
D (1995) a-Scorpion toxins binding on rat brain and insect
sodium channels reveal divergent allosteric modulations by
brevetoxin and veratridine J Biol Chem 270, 15153–15161.
28 Ceste¨le, S & Gordon, D (1998) Depolarization differentially
affects allosteric modulation by neurotoxins of scorpion
alpha-toxin binding on voltage-gated sodium channels J Neurochem.
70, 1217–1226.
29 Little, M.J., Wilson, H., Zappia, C., Ceste¨le, S., Tyler, M.I.,
Martin-Eauclaire, M.-F., Gordon, D & Nicholson, G.M (1998)
d-Atracotoxins from Australian funnel-web spiders compete with
scorpion a-toxin binding on both rat brain and insect sodium
channels FEBS Letters 439, 246–252.
30 Little, M.J., Zappia, C., Gilles, N., Connor, M., Tyler, M.I.,
Martin-Eauclaire, M.-F., Gordon, D & Nicholson, G.M (1998)
d-Atracotoxins from Australian funnel-web spiders compete with
scorpion a-toxin binding but differentially modulate alkaloid toxin
activation of voltage-gated sodium channels J Biol Chem 273,
27076–27083.
31 Zilberberg, N., Gordon, D., Pelhate, M., Adams, M.E., Norris,
F., Zlotkin, E & Gurevitz, M (1996) Functional expression and
genetic modification of an alpha scorpion neurotoxin
Biochem-istry 35, 10215–10222.
32 Kanner, B.I (1978) Active transport of gamma-aminobutyric acid
by membrane vesicles isolated from rat brain Biochemistry 17,
1207–1211.
33 Krimm, I., Gilles, N., Sautiere, P., Stankiewicz, M., Pelhate, M.,
Gordon, D & Lancelin, J.M (1999) NMR structures and activity
of a novel alpha-like toxin from the scorpion Leiurus
Quinques-triatus Hebraeus J Mol Biol 285, 1749–1763.
34 Gordon, D., Moskowitz, H & Zlotkin, E (1990) Sodium channel
polypeptides in central nervous systems of various insects
identi-fied with site directed antibodies Biochim Biophy Acta 1026, 80–
86.
35 Gordon, D., Martin-Eauclaire, M.-F., Ceste¨le, S., Kopeyan, C.,
Carlier, E., Ben-Khalifa, R., Pelhate, M & Rochat, H (1996)
Scorpion toxins affecting sodium channel current inactivation
bind to distinct homologous receptor sites on rat brain and insect
sodium channels J Biol Chem 271, 8034–8045.
36 Gilles, N., Krimm, I., Bouet, F., Froy, O., Gurevitz, M., Lancelin,
J.M & Gordon, D (2000) Structural implications on the
inter-action of scorpion alpha-like toxins with the sodium channel
receptor site inferred from toxin iodination and pH-dependent
binding J Neurochem 75, 1735–1745.
37 Cheng, Y & Prusoff, W (1973) Relationship between inhibition constant (K I ) and the concentration of an inhibitor which causes 50% inhibition (IC 50 ) of an enzymatic reaction Biochem Phar-macol 22, 3099–3108.
38 Weiland, G.A & Molinoff, P.B (1981) Quantitative analysis of drug–receptor interactions I Determination of kinetic and equi-librium properties Life Sciences 29, 313–330.
39 Catterall, W.A (1976) Purification of a toxic protein from scor-pion venom which activates the action potential Na+ionophore.
J Biol Chem 251, 5528–5536.
40 Jover, E., Courand, F & Rochat, H (1980) Two types of scorpion neurotoxins characterized by their binding to two separate receptor sites on rat brain synaptosomes Biochem Biophys Res Comm 95, 1607–1614.
41 Ray, R & Catterall, W.A (1978) Membrane potential dependent binding of scorpion toxin to the action potential sodium iono-phore Studies with a 3-(4-hydroxy 3[ 125 I] iodophenyl) propionyl derivative J Neurochem 31, 397–407.
42 Gilles, N., Leipold, E., Chen, H., Heinemann, S.H & Gordon, D (2001) Effect of depolarization on binding kinetics of scorpion a-toxin highlights conformational changes of rat brain sodium channels Biochemistry 40, 14576–14584.
43 Blaustein, M.P & Goldring, J.M (1975) Membrane potentials in pinched-off presynaptic nerve terminals monitored with a fluo-rescent probe: evidence that synaptosomes have potassium diffu-sion potentials J Physiol 247, 589–615.
44 Ray, R., Morrow, C.S & Catterall, W.A (1978) Binding of scorpion toxin to receptor sites associated with voltage-sensitive sodium channels in synaptic nerve ending particles J Biol Chem.
253, 7307–7313.
45 Catterall, W.A., Morrow, C.S & Hartshorne, R.P (1979) Neu-rotoxin binding to receptor sites associated with voltage-sensitive sodium channels in intact, lysed, and detergent-solubilized brain membranes J Biol Chem 254, 11379–11387.
46 Chen, H., Gordon, D & Heinemann, S.H (2000) Modulation of cloned skeletal muscle sodium channels by the scorpion toxins Lqh-II, Lqh-III, and LqhaIT Pflugers Arch 439, 423–432.
47 Rogers, J.C., Qu, Y., Tanada, T.N., Scheuer, T & Catterall, W.A (1996) Molecular determinants of high affinity binding of a-scor-pion toxin and sea anemone toxin in the S3–S4 extracellular loop
in domain IV of the Na+channel a subunit J Biol Chem 271, 15950–15962.
48 Trainer, V.L., McPhee, J.C., Boutelet-Bochan, H., Baker, C., Scheuer, T., Babin, D., Demoute, J.P., Guedin, D & Catterall, W.A (1997) High affinity binding of pytethrods to the a subunit of brain sodium channels Mol Pharmacol 51, 651–657.
49 Gilles, N., Chen, H., Wilson, H., Legall, F., Montoya, G., Molgo, J., Schnˆherr, R., Nicholson, G., Heinemann, S.H & Gordon, D (2000) Scorpion a- and a-like toxins differentially interact with sodium channels in mammalian CNS and periphery Eur J Neurosci 12, 2823–2832.
50 Smith, R.D & Goldin, A.L (1998) Functional analysis of the rat I sodium channel in xenopus oocytes J Neurosci 18, 811–820.
51 Gordon, D., Merrick, D., Auld, V., Dunn, R., Goldin, A.L., Davidson, N & Catterall, W.A (1987) Tissue-specific expression
of the RI and RII sodium channel subtypes Proc Natl Acad Sci USA 84, 8682–8686.
52 Zilberberg, N., Froy, O., Cestele, S., Loret, E., Arad, D., Gordon, D & Gurevitz, M (1997) Elucidation of the putative toxic-surface of a highly insecticidal scorpion a-neurotoxin affecting voltage-sensitive sodium channels J Biol Chem 272, 14810–14816.
53 Gurevitz, M., Gordon, D., Ben-Natan, S., Turkov, M & Froy, O (2000) Diversification of neurotoxins by C-tail ÔwigglingÕ – a scorpion recipe for survival FASEB J 15, 1201–1205.