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The Journal of Neuroscience, June 1, 2002, 22(11):4364-4371
Domain 2 of Drosophila Para Voltage-Gated Sodium
Channel Confers Insect Properties to a Rat Brain Channel
Iris
Shichor1, 3,
Eliahu
Zlotkin3,
Nitza
Ilan1, 2,
Dodo
Chikashvili1,
Walter
Stuhmer4,
Dalia
Gordon2, and
Ilana
Lotan1
Departments of 1 Physiology and Pharmacology,
Sackler School of Medicine and 2 Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, 69978 Ramat-Aviv,
Israel, 3 Department of Cell and Animal Biology, Institute
of Life Sciences, The Hebrew University, 91904 Jerusalem, Israel, and
4 Max-Planck-Institut fur Experimentelle Medizin, D-37075
Gottingen, Germany
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ABSTRACT |
The ability of the excitatory anti-insect-selective scorpion toxin
AahIT (Androctonus australis hector) to
exclusively bind to and modify the insect voltage-gated sodium channel
(NaCh) makes it a unique tool to unravel the structural differences
between mammalian and insect channels, a prerequisite in the design of selective pesticides. To localize the insect NaCh domain that binds
AahIT, we constructed a chimeric channel composed of rat brain NaCh
 -subunit (rBIIA) in which domain-2 (D2) was replaced by that of
Drosophila Para (paralytic
temperature-sensitive). The choice of D2 was dictated by the
similarity between AahIT and scorpion  -toxins pertaining to both
their binding and action and the essential role of D2 in the -toxins
binding site on mammalian channels. Expression of the chimera
rBIIA-ParaD2 in Xenopus oocytes gave rise to
voltage-gated and TTX-sensitive NaChs that, like rBIIA, were sensitive
to scorpion -toxins and regulated by the auxiliary subunit
1 but not by the insect TipE. Notably, like Drosophila Para/TipE, but unlike
rBIIA/1, the chimera gained sensitivity to AahIT,
indicating that the phyletic selectivity of AahIT is conferred by the
insect NaCh D2. Furthermore, the chimera acquired additional insect
channel properties; its activation was shifted to more positive
potentials, and the effect of -toxins was potentiated. Our results
highlight the key role of D2 in the selective recognition of
anti-insect excitatory toxins and in the modulation of NaCh gating. We
also provide a methodological approach to the study of ion channels
that are difficult to express in model expression systems.
Key words:
Na channel; insect selectivity; Xenopus
oocytes; scorpion toxin; gating; Drosophila Para
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INTRODUCTION |
Voltage gated sodium channels
(NaChs) play a pivotal role in excitability. They are plasma membrane
proteins composed of the large pore-forming -subunit generally
accompanied by one or two auxiliary subunits (Catterall, 1992 ; Feng et
al., 1995). The -subunit is composed of four repeated domains
(D1-D4) consisting of six transmembrane -helixes (S1-S6) and a
pore loop (between S5 and S6) (Catterall, 1995). Many modifiers of
NaChs are used as drugs and insecticides (Kallen et al., 1993; Gordon,
1997a,b). However, being unable to distinguish between NaCh subtypes in
various tissues and species, their use often renders adverse side
effects or toxicity to nontargeted animals. However, some scorpion
neurotoxins are able to exclusively identify insect NaChs (Zlotkin et
al., 1978; Zlotkin, 1999) or distinguish among distinct mammalian
neuronal subtypes (Gilles et al., 1999, 2000). Such selectivity
indicates structural differences at toxin binding sites among NaChs
that, when identified, can be used in the design of selective drugs and insecticides.
Scorpion toxins affecting NaChs bind to two receptor sites on the
-subunit and are divided into - and -classes according to
their mode of action and binding properties (Gordon et al., 1998).
-Toxins inhibit sodium current inactivation during binding to
receptor site-3, which involves extracellular loops S5-S6 of D1 and D4
and S3-S4 of D4 (Thomsen and Catterall, 1989; Rogers et al., 1996; Ma
et al., 2000). -Toxins shift the voltage dependence of activation to
more negative potentials (Wang and Strichartz, 1983) (for review, see
Gordon, 1997b) by binding to receptor site-4 (Jover et al., 1980),
shown to be in domain-2 (D2) (Marcotte et al., 1997; Tsushima et al.,
1999) and to include the external loop S3-S4 (Cestele et al.,
1998).
Two additional groups of scorpion toxins, the excitatory and
depressant, are highly selective to insects (Zlotkin et al., 1978;
Zlotkin, 1997). Like the -toxins acting on mammals, the excitatory
and depressant toxins modify the activation of insect NaChs and compete
for binding with other -toxins (for review, see Gordon et al.,
1998), properties that suggest their affiliation to the -toxin
class. The exclusive recognition of insect NaChs was best demonstrated
using the excitatory toxin from Androctonus australis hector
(AahIT). AahIT is composed of 70 amino acids cross-linked by four
disulfide bridges (Zlotkin et al., 1978) and modifies the gating
mechanism by binding to an external receptor site of insect NaChs
(Pelhate and Zlotkin, 1981, 1982; Gordon et al., 1984, 1992). Its
strict selectivity for insects, which has been documented by toxicity
and electrophysiological and binding experiments (Gordon, 1997a;
Zlotkin, 1997), makes AahIT a unique tool to study the basis of
selective recognition of insect channels.
To clarify the basis of the selective interaction of AahIT with insect
NaChs, aiming to localize the region that is targeted by AahIT, we
constructed a chimeric mammalian-insect channel of the rat brain NaCh
IIA (rBIIA) in which D2 was replaced with that of the Para
(paralytic temperature-sensitive) Drosophila channel. The
chimeric channel was functional, despite the large phylogenetic distance between insects and mammals, and acquired the sensitivity to
the anti-insect selective toxin AahIT.
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MATERIALS AND METHODS |
Materials
Para NaCh DNA was a kind gift from Dr. J. Warmke
(Merck, Rahway, NJ). The clone of TipE was a kind
gift from Dr. M. Williamson (IACR-Rothamsted, Harpenden, UK). AahIT was
purified by column chromatography from the crude venom of the North
African scorpion Androctonus australis hector as described
previously (Zlotkin et al., 1971). LqhIT, Lqh-II (Ltx001) (from
Leiurus quinquestratus hebraeus) was purchased from
Latoxan (Valence, France). TTX was purchased from Sigma (Jerusalem,
Israel). All reagents used were of molecular biology grade.
Construction of a chimeric cDNA, rBIIA-ParaD2
The rat brain NaCh -subunit cDNA (rBIIA, pVA2580) (Gershon et
al., 1992) was restricted by XbaI and BglII to
excise a 1233 bp fragment corresponding to D2 and was isolated by gel
electrophoresis. The 1380 bp parallel fragment of Drosophila Para
cDNA corresponding to D2 was amplified by PCR using
Para Drosophila cDNA as a template and two primers
containing either XbaI or BglII restrictions
sites corresponding to those of the rat brain cDNA: primer 1 (sense), 5'- GCCTCCCGGGGGTCGTATACCTCACATGGCGATCTACTCGGC- '3; and primer 2 (antisense), 5'-GGACAGATCTTCCAGTTGCGTCTGCTCCTTGATCCC-'3.
The PCR conditions were as follows: 27 cycles of 96°C for 1 min,
62°C for 45 sec, and 72°C for 2 min, using the PFU DNA polymerase (Stratagene, La Jolla, CA). The 1380 bp PCR product cDNA was restricted with XbaI and BglII, isolated by agarose gel
electrophoresis, extracted by gel purification kit (QIAEX; Qiagen,
Hilden, Germany), and ligated (T4 DNA ligase; New England
Biolabs, Beverly, MA) to the excised rat brain NaCh cDNA lacking
D2. The ligation products were transformed into competent bacteria
cells (Escherichia coli; JM-109; Promega, Madison,
WI). Colonies were examined by restriction analysis, and
relevant ones were sequenced (automated sequencer; core facilities at
Tel-Aviv University).
Generation of cRNA, injection into Xenopus oocytes,
and electrophysiology
rBIIA cRNA was generated from the pVA2580 construct, linearized
by ClaI, and transcribed in vitro with T7 RNA
polymerase as described previously (Dascal and Lotan, 1992; Gershon et
al., 1992). Chimeric cRNA -subunit was generated from the
rBIIA-ParaD2 cDNA, linearized by PacI, and transcribed with
T7 RNA polymerase. 1-Subunit cRNA was
linearized by NotI and transcribed in vitro with
T7 RNA polymerase (Wallner et al., 1993).
Para and TipE cRNAs were generated from the
pGH19-13-5 Para (Warmke et al., 1997) and pGHTipE (Vais et
al., 2000) constructs, respectively, linearized by NotI, and
transcribed with T7 RNA polymerase. Oocytes were injected with 0.3-0.8
ng of rBIIA cRNA or with 4-10 ng of rBIIA-ParaD2 cRNA with or without
1 cRNA (1:1 ratio) (Catterall, 1995; Makita et
al., 1996) or with 10 ng of Para cRNA with TipE
(1:1 ratio) (Warmke et al., 1997; Vais et al., 2000). Injected oocytes
were incubated at 22°C for 3-4 d in ND96 solution (in
mM: 96 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES, pH 7.5) supplemented with 1.8 mM CaCl2, 2.5 mM sodium pyruvate, and 100 µg/ml
gentamicin, as described previously (Gilles et al., 1999).
Sodium currents were recorded using a Warner Instruments (Hamden, CT)
OC-725B two-electrode voltage-clamp amplifier with a series resistance
compensation circuit and low-resistance agarose-plugged electrodes
filled with 3 M KCl (0.2-0.5 M ). Experiments were done
in a 150 µl bath in ND96 solution supplemented with 1 mM CaCl2, pH 7.6, at 20-22°C. Sodium currents were measured in single oocytes before and after application of the relevant toxin. Toxins were
kept in concentrated stock solutions and were diluted before the
experiment in ND96 solution that contained 1 mg/ml BSA. The application
of the toxin-BSA solution was done directly into the bath, in a volume
up to 15 µl (10% of the bath volume) to get the desired total
concentration. To discard any application artifacts, in few oocytes, 1 mg/ml BSA solution was applied before the application of the toxin.
Before impaling the oocyte with electrodes, the voltage base line was
set to 0 mV. At the end of each experiment, the base line voltage was
checked not to exceed ±2 mV, otherwise the experiment was discarded.
The currents were filtered at 4 kHz and sampled at 11 kHz. Stimulation
and data acquisition were done with an IBM computer using the pClamp
software (Axon Instruments, Foster City, CA). Net current was estimated
by subtraction of scaled leak current.
Data analysis
Voltage dependence of activation and inactivation.
Current-voltage relationship (I-V) data and its
transforming to activation curve, i.e.,
G/Gmax versus
Vtest, were fitted using a nonlinear least-square algorithm, to the modified Bolzmann equation (as by Dascal
and Lotan, 1991) as follows:
G/Gmax = 1/{1 + exp[ (Vtest V1/2)/k]}3
(Eq. 1), where at each membrane voltage
(Vtest) [G = I/(Vtest Vrev)], I is the peak
sodium current, Vtest is the test
potential, and Vrev is the reversal
potential. The three free parameters were
V1/2 (the voltage at which the
probability of a single gate opening is one-half), k (the
slope factor, which corresponds to a change in voltage that produces an
e-fold change in conductance), and
Gmax (the maximal
Na+ conductance attained at very positive voltages).
Steady-state inactivation was studied by holding the oocyte at 80 mV
and stepping the voltage for 200 msec to various values from 90 mV up
to 10 mV in 10 mV increments
(Vprepulse), before measuring
INa+ by a 50 msec voltage step that
elicits maximal current (10 mV for the wild-type rBIIA or 0 mV for
the rBIIA-ParaD2). Fractional current
(I/Imax) was plotted as a
function of Vprepulse and fitted (as
by Dascal and Lotan, 1991) to the following equation: I/Imax = 1/{1 + exp[(Vprepulse V1/2)/k]} (Eq. 2).
Imax was the current obtained by a
prepulse to 90 mV, and all other parameters was as above.
Analysis of the effect of -toxins. Two
parameters were checked to analyze the effect of the -toxins. The
first was the time constant of inactivation of the current described by
the sum of two exponents (a fast component  1
and a slow component 2) and calculated in the
same oocyte before and after application of the toxin. The second
parameter was the estimated affinity (kD) of the
toxin to the channel, determined by the fraction of the maximal peak
current (elicited at 10 or 0 mV for rBIIA or rBIIA-ParaD2, respectively) remaining 5 msec after the peak, before, and 13 min after
application of the toxin. Because the fraction of conductance remaining
5 msec after the peak is proportional to the number of channels
modified by -toxins (Rogers et al., 1996), it can be used to
estimate receptor occupancy and toxin affinity according to the
following formula: KD = [ toxin]
(FG'/FG 1) (Eq. 3), where FG is the
fraction of Na+ current remaining 5 msec
after beginning of the pulse, and FG' is the maximum fraction of current 5 msec after the beginning of the
pulse in the presence of a saturating concentration of -toxin.
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RESULTS |
rBIIA is not sensitive to the excitatory insect-selective toxin
AahIT in contrast to Drosophila Para
The rat brain rBIIA (Noda et al., 1986; Auld et al., 1988, 1990)
and the insect Drosophila Para NaChs (Loughney et al., 1989; Warmke et al., 1997) have been functionally expressed in
Xenopus oocytes, and their biophysical properties were well
characterized. We examined the sensitivity of these two channels,
expressed in Xenopus oocytes, to the scorpion anti-insect
excitatory toxin AahIT, which is toxic to Drosophila flies
(PD50 of 2.3 ng/mg) (Zlotkin et al., 1999). AahIT
(3-10 µM) had no effect on
Na+ current mediated by rBIIA, whereas
1-2 µM AahIT increased significantly the
Na+ current mediated by the
Drosophila Para (Fig. 1) (for
detailed characterization, see Fig. 4C). We aimed to confer
AahIT sensitivity to the mammalian rBIIA by insertion of an insect
channel domain from the Drosophila Para. AahIT shares
biophysical and binding properties with other scorpion toxins of the
-class (Gordon et al., 1984, 1998; Zlotkin et al., 1985; De Lima et
al., 1986; Lazdunski et al., 1986; Lee and Adams, 2000) (see
Discussion). Because the receptor binding site for -toxins (site-4)
was localized to D2 on mammalian NaChs (Marcotte et al., 1997; Cestele
et al., 1998), we constructed a chimeric channel rBIIA-ParaD2 formed
from rBIIA (as a mammalian acceptor channel) and D2 of Para
(as a donor of insect channel; see Materials and Methods).
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Figure 1.
rBIIA is not sensitive to the excitatory insect
selective toxin AahIT, in contrast to Drosophila Para.
Oocytes expressing the Drosophila Para channel (together
with the TipE subunit; A) or the rBIIA channel (together
with the 1-subunit; B) were voltage
clamped at a holding potential of 80 mV, and currents were elicited
by a depolarizing pulse to 10 mV. Na+ currents in
single oocytes were recorded before (control) and
13 min after application of 1 µM
(A) or 3 µM
(B) AahIT.
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Biophysical characterization of the chimeric
channel rBIIA-ParaD2
Expression of the chimeric -subunit rBIIA-ParaD2 in
Xenopus oocytes gave rise to voltage-gated and TTX-sensitive
Na+ currents (Fig.
2A,B),
indicating that NaCh activity was not disrupted by the insertion of the
insect D2. Like rBIIA (Kontis and Goldin, 1993), the chimeric currents
were modulated by coexpression of the mammalian auxiliary
1-subunit; namely, the inactivation was faster
and the amplitudes increased (Isom et al., 1994; Makita et al., 1996)
(Fig. 2A,B). This result indicates
that the interaction between the mammalian - and 1-subunits was
not interrupted by the insect D2, in concert with the documented
involvement of D4 in this interaction (Qu et al., 1999). Notably,
coexpression of the insect auxiliary subunit TipE, required for the
functional expression of the Drosophila Para NaCh
-subunit in Xenopus oocytes (Feng et al., 1995; Warmke et
al., 1997), had no effect on the chimeric channel activity (data not
shown). Thus, all subsequent electrophysiological experiments were
performed with coexpressed - and
1-subunits. To obtain the same amplitudes of
Na+ currents, 10-fold higher cRNA
concentration of rBIIA-ParaD2 was required compared with that of rBIIA
(see Materials and Methods). Expression of both proteins in the
reticulocyte lysate system (Jing et al., 1999) revealed that rBIIA
protein level of expression was ~10-fold higher than that of
rBIIA-ParaD2 (data not shown), suggesting that, at least partly,
impaired expression of rBIIA-ParaD2 is responsible for the low current
amplitudes.
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Figure 2.
Comparison between the chimera,
rBIIA-ParaD2, and the rBIIA currents. A,
B, Top, A schematic presentation of rBIIA
(A) and rBIIA-ParaD2 (B)
channel domains (D1-D4), the latter having rBIIA D1, D3, and D4 and
the Para Drosophila D2 (ParaD2; gray
box). Bottom, Na+ currents
elicited in oocytes expressing -subunits of rBIIA
(A) or rBIIA-ParaD2 (B)
alone () or together with 1- subunit
(+1; 1:1) as denoted.
Oocytes were held at 100 mV, followed by 40 msec depolarizations to
voltages varying from 80 to +30 mV in 10 mV increments. Middle
trace in B shows rBIIA-ParaD2 currents elicited
by stepping to 0 mV from 80 mV holding potential, before and 4 or 8 min after 1 µM TTX application. C,
Normalized peak Na+ currents
(I/Imax) of rBIIA
(open circles) and rBIIA-ParaD2 (filled
circles) plotted as a function of membrane voltage
(V). Each value is mean ± SEM from
seven oocytes. The experimental results were fitted using a modified
Bolzmann equation (Eq. 1 in Materials and Methods). D,
Normalized conductance
(G/Gmax)-voltage
relationships derived from the data in C. Data were
fitted using Equation 1 (see Materials and Methods).
Symbols are as in C. Values for
half-activation potential (V1/2) and
for the slope factor (k) were as follows: rBIIA,
V1/2 = 24.7 ± 1.4 mV,
k = 2.3 ± 0.24 mV; rBIIA-ParaD2,
V1/2 = 13.6 ± 0.8 mV,
k = 5.1 ± 0.56 mV; showing a significant
difference between rBIIA and rBIIA-ParaD2 (paired two-tailed
t test; p < 0.001).
E, Steady-state inactivation of the rBIIA
(open circles) and rBIIA-ParaD2 (filled
circles). Oocytes were held at 80 mV, and 200 msec steps to
prepulse potentials (Vprepulse) from 90 to
10 mV in 10 mV increments were given before eliciting currents by 50 msec steps to 10 or 0 mV (as detailed in Materials and Methods).
Fractional current
(I/Imax) was plotted
as a function of Vprepulse and fitted to
Equation 2 (see Materials and Methods). Each point
represents mean ± SEM values from four oocytes. Values for
half-inactivation potential (V1/2)
and for the slope factor (k) were as follows:
rBIIA, V1/2 = 49 ± 0.75 mV,
k = 5.7 ± 0.12 mV; rBIIA-ParaD2,
V1/2 = 54 ± 0.7 mV,
k = 6.4 ± 0.13 mV; showing a significant
difference between rBIIA and rBIIA-ParaD2 (using paired two-tailed
t test; p < 0.005 for
V1/2; p < 0.01 for k).
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Comparison between rBIIA and rBIIA-ParaD2 biophysical characteristics
revealed a positive shift of +11 mV in the half-activation voltage
(V1/2) of the chimera, accompanied by
an increase of 2.8 mV in the slope factor (k), compared with
rBIIA (Fig. 2C,D), and both effects were
statistically highly significant (p < 0.001). The activation characteristics of the rBIIA-ParaD2
(V1/2 = 13 mV; k = 5.1 mV) (Fig. 2D) were similar to those of the Para
Drosophila Na+ channel, which
was previously fully characterized in oocytes (Warmke et al.,
1997). The -subunit of the Para Drosophila, expressed with TipE, was shown to have V1/2 = 16.9 mV and k = 5.43 mV. Thus, it seems that the
rBIIA-ParaD2 chimera acquired insect channel properties related to
voltage-dependent activation, which is in concert with the importance
of D2 in the voltage sensitivity of the NaChs (Auld et al., 1990;
Marcotte et al., 1997; Qu et al., 1999). The steady-state inactivation
characteristics of the rBIIA-ParaD2 were slightly different, but
statistically significant, from those of rBIIA (Fig.
2E) and were within the range of values determined for the Drosophila Para channel expressed in
Xenopus oocytes (Warmke et al., 1997).
rBIIA-ParaD2 is sensitive to AahIT
Comparison of the effects of AahIT on rBIIA and rBIIA-ParaD2
channels (Fig. 3) revealed that 3 µM AahIT had no effect on rBIIA currents at all test
potentials, as shown in the I-V relationship (Fig.
3A, bottom). In contrast, 1.4 µM AahIT increased markedly the rBIIA-ParaD2
current, as shown for currents elicited at 30 and 20 mV (Fig.
3B, top). Analysis of the voltage dependence of
activation of the rBIIA-ParaD2 revealed a statistically significant (p  0.003) negative shift of 6 mV in
V1/2 in the presence of AahIT (Fig.
3B, middle and bottom). This effect
resembles the negative shift in the voltage dependence of activation of
Na+ currents in insect neurons induced by
AahIT (Lee and Adams, 2000). As described previously (Marcotte et al.,
1997; Cestele et al., 1998; Tsushima et al., 1999), such an effect is
typical to -toxins acting on mammalian
Na+ channels.
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Figure 3.
AahIT affects the rBIIA-ParaD2 but not the rBIIA
channels. A, B, Top, rBIIA
(A) and rBIIA-ParaD2 (B)
currents elicited after step depolarizations to 30 or 20 mV (as
denoted above traces) before and 10 min after
application of 3 µM (A) or 1.4 µM (B) AahIT. Concentrations up to
10 µM were checked to give the same results.
Middle, Normalized peak currents
(I/Imax) of each
oocyte plotted as a function of membrane voltage
(V), before (open circles,
control) and 10 min after (filled
circles) application of AahIT. Each point
represents the mean ± SEM values from three oocytes treated with
3 µM (A) or of six oocytes treated
with 1.4-2.8 µM (B) AahIT. Data
were fitted using Equation 1 (see Materials and Methods). No
significant difference between maximal currents of control and
AahIT-treated oocytes was observed. Bottom, Normalized
conductance
(G/Gmax)-voltage
relationships derived from the data in the middle panel,
fitted to Equation 1. Values for V1/2 and
k were as follows: control,
V1/2 = 10 ± 1.2 mV,
k = 4.7 ± 0.15 mV; AahIT-treated oocytes,
V1/2 = 15.5 ± 1 mV,
k = 4.99 ± 0.07 mV; showing a significant
difference (p 0.003 using two-tailed
paired t test) in V1/2
between the control and AahIT.
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Conditioning pulse potentiates the effect of AahIT on the
rBIIA-ParaD2 channel
Depolarizing conditioning pulse was demonstrated to be crucial for
the activity of the -toxin Css-IV (from Centruroides
suffusus suffusus) on the rat brain NaCh rBIIA (Cestele et al.,
1998) or to potentiate the effect of other -toxins on mammalian
NaChs (Tsushima et al., 1999). Based on the results showing that AahIT belongs to the -toxin class (Fig. 3), we wanted to examine the effect of depolarizing conditioning pulse on the activity of AahIT. To
this end, we compared in single oocytes the effect of AahIT with and
without a 2 msec depolarizing prepulse to +50 mV (Cestele et al.,
1998). The results obtained in a representative oocyte clearly show
that the prepulse potentiated the effect of AahIT on rBIIA-ParaD2
currents (Fig. 4A,
compare prepulse with + prepulse). With
the prepulse, AahIT not only shifted the voltage dependence of
activation but also increased the maximal current
(Imax). Steady-state activation
analysis in several oocytes (Fig. 4B) not only
revealed a prepulse-independent shift in the equilibrium activation
potential (~5.5 mV) but also an increase in the maximal conductance
(Gmax) with the prepulse by 23 ± 6% (n = 4; p 0.033). Thus, using
the prepulse, we further revealed the similarity between the effects of
AahIT on rBIIA-ParaD2 and that of -toxins on mammalian NaChs.
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Figure 4.
The effect of AahIT is potentiated by a
conditioning depolarizing pulse. A, rBIIA-ParaD2
currents elicited by step depolarization to 10 mV without ( prepulse, left) or after (+ prepulse, middle) a 2 msec prepulse to
+50 mV, before (control) and after
(AahIT) application of 1.4 µM
AahIT. Right, Normalized rBIIA-ParaD2 current-voltage
relationships measured in a single oocyte, before (open
triangles, control) and after
(filled triangles) application of 1.4 µM AahIT using the prepulse protocol. Data showing the
effect of the toxin in the same oocyte without using the prepulse
protocol (as in Fig. 3B) was superimposed
(open and filled circles for control and
AahIT, respectively). Currents were normalized to maximal current of
control. Data were fitted using Equation 1 (see Materials and Methods).
Gmax values without prepulse were as
follows: 53.2 and 52.4 µS for control and AahIT-treated oocytes,
respectively. Gmax values with prepulse were
as follows: 43.5 and 50.6 µS for control and AahIT-treated oocytes,
respectively. B, C, Left,
Normalized peak currents
(I/Imax of control) of
rBIIA-ParaD2 (B) or Para/TipE
(C) plotted as a function of membrane voltage
(V), before (open
triangles) and 10 min after (filled
triangles) application of 1-2 µM AahIT, using a
prepulse protocol. Each point represents the mean ± SEM values from four oocytes. Right, Voltage
dependence of activation derived from the current-voltage
relationships (left) presented as an activation curve of
control (open triangles) and AahIT-treated
(filled triangles) oocytes. Normalized
G-V relationships were fitted using Equation 1 (see
Materials and Methods). Values for rBIIA-ParaD2
(B) were as follows: control,
V1/2 = 14 ± 0.67 mV,
k = 4.7 ± 0.09 mV,
Gmax = 31.48 ± 3; AahIT-treated
oocytes, V1/2 = 19.5 ± 1 mV,
k = 4.6 ± 0.1 mV,
Gmax= 38.6 ± 2.6; showing significant
differences (paired two-tailed t test) in
V1/2 (p < 0.005)
and Gmax (p < 0.033) between the control and AahIT-treated oocytes. Values for
Para/TipE (C) were as follows:
V1/2 = 19 ± 2.23 , k = 6.5 ± 0.3, Gmax = 19.42 ± 1.37;
AahIT-treated oocytes, V1/2 = 22.2 ± 2.7, k = 6.2 ± 0.2, Gmax = 26.31 ± 2.84; showing
significant differences (paired two-tailed t test) in
V1/2 (p < 0.007)
and Gmax (p < 0.019) between the control and AahIT-treated oocytes. D,
Normalized currents
(I/Imax), elicited
using the prepulse protocol, plotted as a function of membrane voltage
(V) in either control (open
triangles; n = 7) or 10 µM
AahIT treated (filled triangles;
n = 4) in rBIIA-expressing oocytes.
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Analysis of the effect of 1-2 µM AahIT on the
Drosophila Para (the donor of D2) coexpressed with TipE
revealed an increase in the maximal conductance by 35 ± 10%
(n = 4; p 0.019) and a shift in the
equilibrium activation potential of ~3 mV, the former effect being
larger and the latter effect being smaller than those observed in the
chimera (Fig. 4C). Notably, the rBIIA channel remained
insensitive to AahIT also with the prepulse (Fig. 4D).
Effects of scorpion -toxins on the rBIIA-ParaD2 channel
Because rBIIA-ParaD2 was found to be sensitive to the
insect-selective toxin AahIT, we wanted to examine whether this
insect-mammalian chimera also acquired sensitivity to the -toxin
LqhIT, which is highly active on insects (Eitan et al., 1990).
rBIIA-ParaD2 was poorly sensitive to LqhIT; the small decrease in
2 (Fig. 5A, Table
1), however, may be attributed to
modifications of intrinsic gating properties rather than to a change in
toxin sensitivity caused by the insertion of the insect D2 (Fig. 2)
(see below). The rat brain channel rBIIA was practically insensitive to
LqhIT, even at concentrations as high as 5 µM (Fig. 5A, Table 1), as expected
(Gilles et al., 1999). To rule out the possibility that the chimera
became resistant to -toxins in general, we tested the effect of the
anti-mammalian -toxin Lqh-II (Sautiere et al., 1998; Gilles et al.,
1999, 2000). Lqh-II caused a typical inhibitory effect on the
inactivation of both rBIIA and rBIIA-ParaD2 currents (Fig.
5B) (Rogers et al., 1996; Gilles et al., 1999, 2001; Chen et
al., 2000). The KD values (a measure
of the apparent affinity of the toxin for its receptor) of Lqh-II to
the two channels were estimated by measuring the fraction of the
current remaining 5 msec after the beginning of the pulse in the
absence and presence of the toxin (see Materials and Methods). The
estimated KD values for both channels
(12.78 ± 4.2 nM, n = 5;
6.26 ± 1.76 nM, n = 5; for
rBIIA and rBIIA-ParaD2, respectively) were found not to differ
statistically (p > 0.05), suggesting that the
change in D2 had no major effect on the binding site of the -toxin
Lqh-II. Analysis of the effect of Lqh-II on the time constants of
inactivation showed a significant increase in the fast (1) and slow
(2) constants of both rBIIA and rBIIA-ParaD2 channels (Table 1).
Notably, the slowing of channel inactivation by Lqh-II was more
profound in the rBIIA-ParaD2 channel chimera compared with rBIIA (Table
1, Fig. 5). Moreover, the potentiated effect of Lqh-II on rBIIA-ParaD2 was also manifested by the larger increase in current amplitude [by
3.6 ± 0.6 (mean ± SEM) and 1.76 ± 0.09, for
rBIIA-ParaD2 and rBIIA, respectively] (Fig. 5B). These
results resemble those obtained with another site-3 toxin from sea
anemone (ATXII) on Drosophila Para NaCh expressed in oocytes
(Warmke et al., 1997). Despite having comparable affinities to rBIIA
and to Drosophila Para, ATXII had a larger effect on the
insect channel, manifested in both larger increase in maximal
conductance and larger slowing of inactivation. We also verified that
20 nM LqhIT practically abolished inactivation
and caused a marked increase in the maximal conductance (data not
shown), similar to the effect of ATXII on this insect channel.
Together, the high sensitivity of the Para channel, as opposed to the
resistance of the rBIIA-ParaD2, to LqhIT and the strong effect of
Lqh-II on both rBIIA and the chimera indicate that D2 neither
contribute to the binding of receptor site-3 or to the selectivity of
-toxins. On the other hand, the larger effects of Lqh-II on the
chimeric channel indicate that D2 conferred insect properties related
to both activation and inactivation gating.
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Figure 5.
The effect of scorpion -toxins on rBIIA-ParaD2
and rBIIA. Oocytes were held at 80 mV, and maximal rBIIA or
rBIIA-ParaD2 currents, recorded every 1 min, were elicited by a
depolarizing pulse to 10 or 0 mV, respectively. A,
rBIIA-ParaD2 (right) and rBIIA (left)
currents, before (control) and 13 min after
application of 1 µM LqhIT. Same results were obtained
with the toxin at 5 µM. B, rBIIA-ParaD2
(right) and rBIIA (left) currents, before
(control) and 13 min after application of 200 nM Lqh-II.
|
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DISCUSSION |
In this study, using the insect-selective neurotoxin AahIT, we
provide a first step toward deciphering the structural elements that
are responsible for the selective recognition of insect versus mammalian NaChs by drugs and toxins. With the background of the close
similarity in the primary structure, topological organization, and
basic biochemical and pharmacological properties between the mammalian
and the insect NaChs (Gordon, 1997a), we used a mammalian-insect chimeric channel to identify the region that is recognized by AahIT and
is responsible for its selective insect toxicity.
The choice of the chimera
The Drosophila Para NaCh, which is sensitive to AahIT
(Figs. 1A, 4C), was chosen as a donor of
an insect channel region that will confer AahIT sensitivity to the
chimeric channel. The rat brain NaCh rBIIA, shown to be insensitive to
high doses of AahIT (Fig. 1B), was chosen as the
mammalian background. Both NaChs have been well characterized in
Xenopus oocytes. To choose the appropriate region of the
insect channel, which would confer the sensitivity to AahIT, we
considered the following line of evidence that led us to assign AahIT
to the -toxin class, the receptor site of which is comprised mainly
of a single channel domain, D2 (see introductory remarks): (1) like
scorpion -toxins active on mammals (Cahalan, 1975; Jaimovich et al.,
1982; Wang and Strichartz, 1983; Vijverberg et al., 1984; Jonas et al.,
1986), AahIT modified the activation process of sodium currents, as
shown in cockroach axon (Zlotkin et al., 1985) and in lepidopterous
(Heliothis virescens) neurons (Lee and Adams, 2000); (2)
AahIT competed with the binding of the -toxin Ts-VII (or  -toxin,
from Tityus serrulatus) (Lazdunski et al., 1986) on
cockroach neuronal membranes (De Lima et al., 1986); and (3) like
-toxins binding to mammalian NaChs (Jover et al., 1980; Lazdunski et
al., 1986), AahIT binding to insect (Locusta migratoria)
neuronal membranes was not modified by membrane potential or by the
alkaloid toxin veratridine (Gordon et al., 1984). Having assigned AahIT
to the -class of neurotoxins, we assumed that, like on mammalian
channels (Marcotte et al., 1997; Cestele et al., 1998; Tsushima et al.,
1999), the -toxin site on insect channels also resides in D2 and
thereby is targeted by AahIT. This assumed that homology between toxin
receptor sites on insect and mammalian NaChs was supported by the
corresponding homology demonstrated for the -toxin receptor sites
(Gordon and Zlotkin, 1993; Gordon et al., 1996, 2002; Gilles et al.,
1999). Thus, we constructed the chimeric channel rBIIA-ParaD2
consisting of rBIIA and D2 of the insect Para
Drosophila.
Functional NaCh integrity of the rBIIA-ParaD2 chimera
rBIIA-ParaD2 formed a functional, voltage-dependent and
TTX-sensitive NaCh (Fig. 2B), despite the large
phylogenetic distance between flies and rodents. rBIIA-ParaD2 was also
regulated by the coexpressed 1-subunit (Fig.
2A,B), consistent with the finding that the binding site for 1 was localized to
D4 in rBIIA (Qu et al., 1999). The chimeric channel was, however, not
sensitive to the coexpressed insect auxiliary subunit TipE, excluding
the involvement of D2 in the interaction of the insect channel
-subunit with TipE. Scorpion -toxin Lqh-II was used to further
assess the functional NaCh integrity of the chimeric channel. The
binding of -toxins to an extracellular receptor site (see
introductory remarks) affects fast inactivation, a process that
involves internal channel regions (Stuhmer et al., 1989; Patton et al.,
1992); thus, the toxin effect may reflect a chain of intermolecular
changes (Catterall, 1992; Gordon, 1997b; Gilles et al., 2001). The fact that Lqh-II affected the inactivation of the chimeric channel (Fig.
5B) ensured that the overall assembly and activity of the channel were not impaired. This functional NaCh integrity provided a
suitable setting for the study of insect characteristics, including AahIT sensitivity, that were gained by the chimera.
Domain 2 of the insect channel confers NaCh sensitivity
to AahIT
Two apparent effects on Na+ current
activation were documented for the anti-insect toxin AahIT in neuronal
preparations of various insects: an increase of maximal current and a
negative shift of the voltage dependence of activation (Zlotkin, 1997). Thus, AahIT increased the Na+ peak current
in cockroach axons and shifted the activation curve (Zlotkin, 1997),
whereas, in intact insect neurons (Heliothis virescens,
Lepidoptera), only the shift in the activation was observed (Lee and
Adams, 2000). In the Drosophila (fruit fly) Para NaCh
coexpressed with TipE in oocytes, the main effect of AahIT was an
increase of the peak current (by 35%) accompanied by a small shift
(~3 mV) in the activation curve (Fig. 4C). It appears that
the sensitivity to AahIT varies among the different insect species and
may depend also on channel preparation (native channels in neurons vs
expressed channels in oocytes). Notably, AahIT, at concentrations
similar to those used for Drosophila Para NaCh, exerted both
effects on the rBIIA-ParaD2: an increase in the peak current (by 20%)
and a shift in the activation curve (by ~6 mV) (Fig. 3B).
In view of the similar toxin sensitivity of the chimeric channel to the
insect Para channel, together with the absolute toxin resistance of the
mammalian channel rBIIA (Fig. 1), these results indicate that the
receptor site for the insect-selective toxin AahIT resides mainly in
ParaD2. Also, TipE is apparently not involved in the toxin selectivity
to insect channels, because the chimera was sensitive to AahIT in its absence.
Interestingly, the concentrations of AahIT required to elicit the
effects in neurons (50-100 nM) (Lee and Adams, 2000) were 10-fold lower than those required for the chimeric and
Drosophila Para channels (1-2 µM).
These differences may be attributed to variations in susceptibility of
various insects to the toxin (Fishman et al., 1997), which most
probably vary in their NaCh Para gene homologs. Such
variations may be exemplified by Sarcophaga blowfly larvae,
which are at least 130-fold more sensitive to AahIT than Drosophila melanogaster wild-type flies. Moreover, in
various Drosophila strains, which differ by a few
substitutions in their gene encoding the Para channel, the sensitivity
to AahIT is reduced by up to 2000-fold compared with blowflies (Zlotkin
et al., 1999).
The negative shift in the voltage sensitivity of activation induced by
AahIT on insect and the rBIIA-ParaD2 channels resembles the effect of
scorpion -toxins on mammalian channels (Cahalan, 1975; Meves et al.,
1982; Wang and Strichartz, 1983; Vijverberg et al., 1984; Jonas et al.,
1986; Marcotte et al., 1997; Cestele et al., 1998; Tsushima et al.,
1999). Furthermore, the effects of AahIT on rBIIA-ParaD2 was
potentiated by a depolarizing prepulse (Fig. 4), as was shown
previously for some -toxins (Cestele et al., 1998; Tsushima et al.,
1999), thus establishing the similarity between AahIT and -toxins
modes of action.
The rBIIA-ParaD2 acquired additional insect NaCh properties
In addition to the sensitivity to AahIT, the rBIIA-ParaD2 acquired
inherent activation parameters [a positive shift of the half
activation voltage (V1/2)] (Fig.
2D) that were similar to those of the intact
Drosophila Para channel expressed in oocytes (Warmke et al.,
1997) (Fig. 4C). The critical impact of the origin of D2 on
the voltage-dependent activation of NaChs was demonstrated previously
(Marcotte et al., 1997). In view of the 65% identity between the
entire D2 of rBIIA and Drosophila Para, sharing, however, a
100% identity in their voltage sensors (D2-S4), our results highlight
the importance of structural elements other than D2-S4 in the
activation process of NaChs. Indeed, in addition to the previously
demonstrated critical impact of mutations in D2-S4 (Stuhmer et al.,
1989; Auld et al., 1990; Moran et al., 1994), changes in the external
loops of D2 in mammalian NaChs were also shown to affect the voltage
dependence of activation (Cestele et al., 1998; Qu et al., 1999).
Other insect properties conferred by the insect D2 were revealed by
analyzing the effect of the site-3 -toxin Lqh-II on the chimeric
channel. Lqh-II affected the inactivation of rBIIA-ParaD2 more potently
than that of rBIIA (Fig. 5B, Table 1). This effect was not
accompanied by a change in the toxin binding affinity (Fig. 5),
supporting the notion that D2 does not contribute to receptor site-3
(Gordon et al., 2002). Because D2 has not yet been implicated in any
phenomenon pertaining to NaCh inactivation, our results indicate
involvement of D2 in inactivation, in addition to its expected
involvement in activation (as detailed above).
Additional considerations
Two additional inferences regarding NaChs can be made on the basis
of the results obtained in this study. (1) The findings that the
binding of both -toxins Lqh-II and LqhIT were not affected by the
change in D2 (Fig. 5) indicate that the selectivity of -toxins
toward rat brain and insect NaChs is not conferred by D2. (2) The
insect Para Drosophila NaCh is hardly expressed in oocytes
without the coexpression of the auxiliary subunit TipE (Feng et al.,
1995; Warmke et al., 1997). We show, however, that the mammalian
channel rBIIA can acquire insect channel properties by virtue of
insertion of a single domain of Para Drosophila channel without the need of TipE. Thus, the chimera approach presents a
technological advantage for the study of NaChs in heterologous systems,
which circumvents possible difficulties resulting from missing
additional subunits.
| |
FOOTNOTES |
Received Jan. 17, 2002; revised March 17, 2002; accepted March 12, 2002.
This work was partly supported by Israeli Science Foundation Grants
437/98 (I.L.) and 508/00 (D.G.), United States-Israel Binational
Science Foundation Grant 1999396 (I.L.), and German-Israeli Foundation
Grant I 0375-172 (W.S., E.Z., I.L., and D.G.). We thank Nathan Dascal
for helpful discussions and Jeffrey Warmke and Martin Williamson for
the kind gifts of the Drosophila Para and
TipE clones, respectively.
Correspondence should be addressed to either Prof. Ilana Lotan,
Department of Physiology and Pharmacology, Sackler School of Medicine,
Tel-Aviv University, 69978 Ramat-Aviv, Israel, E-mail: ilotan{at}post.tau.ac.il; or Dr. Dalia Gordon, Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, 69978 Ramat-Aviv, Israel, E-mail: dgordon{at}post.tau.ac.il.
| |
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