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com

articles

Inhibition of T-type voltage-gated

calcium channels by a new
scorpion toxin
Rosalind S-I. Chuang1, Howard Jaffe2, Leanne Cribbs3, Edward Perez-Reyes3 and Kenton J. Swartz1

1 Molecular Physiology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health,
Bldg. 36, Rm. 2C19, 36 Convent Drive, MSC 4066, Bethesda, Maryland 20892, USA
2 Protein/Peptide Sequencing Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health,
Bldg. 36, Rm. 3B26, 36 Convent Drive, MSC 4066, Bethesda, Maryland 20892, USA
3 Department of Physiology, Loyola University Medical Center, 2160 South First Avenue, Maywood, Illinois 60153, USA
© 1998 Nature America Inc. • http://neurosci.nature.com

Correspondence should be addressed to K.J.S. (kjswartz@codon.nih.gov)

The biophysical properties of T-type voltage-gated calcium channels are well suited to pacemaking
and to supporting calcium flux near the resting membrane potential in both excitable and non-
excitable cells. We have identified a new scorpion toxin (kurtoxin) that binds to the α1G T-type
calcium channel with high affinity and inhibits the channel by modifying voltage-dependent gating.
This toxin distinguishes between α1G T-type calcium channels and other types of voltage-gated
calcium channels, including α1A, α1B, α1C and α1E. Like the other α-scorpion toxins to which it is
related, kurtoxin also interacts with voltage-gated sodium channels and slows their inactivation.
Kurtoxin will facilitate characterization of the subunit composition of T-type calcium channels and
help determine their involvement in electrical and biochemical signaling.

The biophysical properties of T-type voltage-gated calcium Results

channels differ from those of other types of voltage-gated cal-
cium channels in that they activate at voltages near the rest-
ing membrane potential of many cells and inactivate (close
despite continued depolarization) rapidly1–8. T-type calcium
channels also deactivate (close) slowly and have a small sin-
gle-channel conductance2–8. Because of these unique gating
properties, T-type calcium channels are thought to be involved
in pacemaking in neurons and cardiac myocytes1,9–11 and to
contribute to a standing calcium current near the resting
membrane potential in various cell types11–13. The expression
of T-type calcium channels outside the nervous system8,12–14
suggests that they have other important functions.
Progress in understanding the subunit composition and
physiological roles of T-type calcium channels has been hin-
dered by a scarcity of ligands that can distinguish between T-
type calcium channels and other types of voltage-gated
calcium channels. T-type calcium channels are not inhibited
by many types of calcium channel inhibitors, and the
inhibitors that do act on T-type calcium channels lack speci-
ficity. For example, mibefradil, perhaps one of the best lig-
ands for T-type calcium channels, also inhibits other types of
high-voltage-activated calcium channels15. We began a search
for a ligand that binds to T-type voltage-gated calcium chan-
nels using the recently identified α 1G T-type calcium chan-
nel 16 as a target. Venoms from different scorpions were
examined for activity against the α1G T-type calcium channel
expressed in Xenopus oocytes. Here we describe the purifica-
tion, biochemical analysis and biophysical characterization of
kurtoxin, a new protein toxin from the venom of a South
African scorpion (Parabuthus transvaalicus).
Expression of the recently cloned α1G calcium channel in
Xenopus oocytes gives rise to calcium channels with bio-
physical characteristics resembling those of native T-type
calcium channels studied in neurons, muscle and other
cell types16. To improve expression, we subcloned the α1G
cDNA into a vector containing the 5’ and 3’ untranslated
regions of a Xenopus β-globin gene17. Injection of oocytes
with cRNA for the α1G channel flanked by these untrans-
lated sequences resulted in robust expression of T-type
channels (peak inward currents of 1 to 10 µA with 5 mM
barium as charge carrier) within 4 to 7 days.
We screened the venom from different scorpions for
activity against the α 1G T-type calcium channel. The
venom of Parabuthus transvaalicus contained activity that
produced robust inhibition of the α1G calcium channel
(not shown). The scorpion venom was fractionated by
reversed-phase high-performance liquid chromatogra-
phy (HPLC), and individual fractions were examined for
activity against the α1G calcium channel. After two con-
secutive separations (Fig. 1a and b), a single symmetrical
peak (Fig. 1c) had strong inhibitory activity. The pri-
mary amino-acid sequence of the active material was
determined by Edman degradation. The protein, which
we named kurtoxin, contains 63 amino-acid residues
(Fig. 1d). The predicted mass of this protein is
7386.5 daltons, consistent with a mass of 7386.1 daltons
measured using mass spectrometry. Comparison of the
amino-acid sequences of kurtoxin and other previously
described protein toxins indicates that kurtoxin is close-
ly related to the α-scorpion toxins, a large family of tox-

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articles

ins that slow inactivation of voltage- a b

Absorbance (280 nm)

Absorbance (215 nm)

gated sodium channels18.
We examined the inhibitory effect of
kurtoxin on α1G T-type calcium channels
expressed in Xenopus oocytes (Fig. 2).
The channel was activated by depolariz-
ing steps to –40 mV from a holding volt-
Time (min) Time (min)
age of –90 mV, given every 10 seconds.
External application of kurtoxin inhibit- c d

Absorbance (215 nm)

ed voltage-activated barium currents in
oocytes expressing the α1G calcium chan-
nel. After removal of the toxin from the
recording chamber, calcium channel cur-
rents recovered slowly (τoff ~250 s).
Because kurtoxin is highly related to
the α-scorpion toxins, which are known
Time (min)
gating modifiers of sodium channels, we
anticipated that kurtoxin was not Fig. 1. Purification of kurtoxin from scorpion venom. (a) Initial fractionation of Parabuthus
© 1998 Nature America Inc. • http://neurosci.nature.com

inhibiting the T-type calcium-channel transvaalicus venom by reversed-phase HPLC. The chromatogram shown resulted from the
by a pore-blocking mechanism, but by injection of the component of venom (0.5 mg) soluble in 25% aqueous acetonitrile with 0.1%
modifying channel gating. We examined trifluoroacetic acid. The asterisk indicates the peak containing kurtoxin. The solvent system
this possibility by studying the effects of used aqueous acetonitrile with trifluoroacetic acid. (see Methods) (b) The active peak in (a),
kurtoxin at different voltages (Fig. 3). At indicated by the asterisk, was fractioned a second time using reversed-phase HPLC yielding
a concentration of 350 nM, kurtoxin pure kurtoxin as indicated. The solvent system used aqueous methanol with trifluoroacetic
inhibited T-type calcium-channel cur- acid (see Methods). (c) Chromatogram of pure kurtoxin. Chromatography was the same as
rent elicited by a weak depolarization by in (b). (d) Complete primary amino-acid sequence of kurtoxin.
over 95% (Fig. 3a). However, calcium-
channel currents elicited by strong depo-
larization were less inhibited by kurtoxin
(Fig. 3a). Activation curves for the α 1G calcium channel macroscopic and single-channel currents suggested that the
(Fig. 3b) showed that 350 nM kurtoxin shifted the opening of inactivation step for T-type calcium channels is not intrinsi-
the α1G calcium channel to more positive voltages and that the cally voltage dependent, but derives its apparent voltage depen-
slope of the activation–voltage relation was more shallow in dence at negative voltages from coupling to voltage-dependent
the presence of the toxin. These effects of kurtoxin are similar activation steps8. The convergence of τinactivation at depolarized
to what has been observed for grammotoxin19, ω-AgaIVA 20 voltages (Fig. 4c) suggests that the effect of kurtoxin on inac-
and hanatoxin21, three established gating modifiers of other tivation kinetics is probably secondary to an effect of the toxin
types of voltage-gated ion channels. The α1G calcium channels on transitions in the activation pathway. These results, togeth-
opened by large depolarization in the presence of kurtoxin er with the sequence homology between kurtoxin and other
deactivated with kinetics virtually identical to those of the con- known gating-modifier toxins (for example, α-scorpion tox-
trol channels without toxin (Fig. 3c). In contrast, the activa-
tion kinetics of channels opened by strong depolarization in
the presence of kurtoxin were much slower than those of con- a
trol channels (Fig. 3c, see also Fig. 4b). The effects of kurtoxin
on inactivation were examined using longer (50 ms) depolar-
izing pulses (Fig. 4). Channels opened by moderate depolar-
ization (for example, 0 mV) in the presence of toxin inactivated
more slowly (τ = 25 ms) than control channels (τ = 6 ms) at
the same voltage (Fig. 4b). The slowed inactivation for channels 10 ms
opened in the presence of toxin became less pronounced with
stronger depolarization (Fig. 4b and c; at +100 mV, τ = 8 ms
in control and τ = 12 ms in toxin). Previous analyses of both

b
Fig. 2. Inhibition of α1G T-type calcium channels by kurtoxin.
(a) Currents elicited by 30 ms depolarizations to –40 mV in the
absence or presence (200 nM) of kurtoxin. Charge carrier was 5
IBa2+ (µA)

mM barium. Holding voltage was –90 mV, and tail voltage was –60
mV. Dashed lines indicate zero current. Linear capacitive current
and very small background conductances, isolated by blocking cal-
cium channels with 1 mM CdCl2, have been subtracted. (b) Time
course for both onset and recovery from inhibition by kurtoxin.
Peak inward barium current is plotted against time. The pulse pro-
tocol illustrated in (a) was repeated every 10 s. Time (s)

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articles

a b c

Itail (µA)
10 ms
Test voltage (mV)
10 ms

Fig. 3. Kurtoxin shifts the opening of the α1G T-type calcium channel to more depolarized voltages. (a) Currents elicited by 7.5-ms depolar-
izations to various voltages in the absence (open circles) or presence (closed circles; 350 nM) of kurtoxin. Holding voltage was –90 mV, and
tail voltage was –60 mV. Dashed lines indicate zero current. Linear capacitive current and very small background conductances have been
subtracted using 1 mM CdCl2. (b) Activation curves in the absence (open circles) and presence (closed circles; 350 nM) of kurtoxin. Tail
amplitude, measured 1 ms after repolarization to –60 mV, plotted against test voltage. Holding voltage was –90 mV. Linear capacitive current
© 1998 Nature America Inc. • http://neurosci.nature.com

and very small background conductances have been subtracted using 1 mM CdCl 2. In control solution, the Itail–voltage relation at a constant
test-pulse duration of 7.5 ms does not precisely reflect the voltage dependence of steady-state activation. At negative voltages, 7.5 ms is not
quite long enough to reach steady state, and at positive voltages, many channels inactivate before the tail current is elicited (see traces in a).
(c) Scaled traces for depolarizations to +80 mV in either control or 350 nM kurtoxin. Tail voltage was –60 mV.

ins), suggest that kurtoxin inhibits the α 1G T-type calcium
channel by modifying the energetics of channel gating.
We examined the concentration dependence for inhibition
of the α1G calcium channel by kurtoxin. The fraction of unin-
hibited current does not faithfully approximate the fraction
of unbound channels at all voltages because kurtoxin shifts
the activation of channels to more depolarized voltages. We
therefore used the approach described for hanatoxin 21 , in
a ited current reached a plateau at negative voltages and
10 ms
b c
which the fraction of uninhibited current at negative voltages
was used to estimate the fraction of unbound channels.
(Because kurtoxin does not affect deactivation kinetics, we
cannot use tail-current kinetics to directly distinguish between
the opening of toxin-unbound and toxin-bound channels, as
has been done with hanatoxin21). Current amplitudes were
measured following various weak depolarizations in the pres-
ence of different concentrations of kurtoxin (Fig. 5a). Con-
sistent with previous observations for hanatoxin inhibition of
a voltage-gated potassium channel21, the fraction of uninhib-
increased with stronger depolarization. The fraction of unin-
hibited current at negative voltages, which approximates the
fractional occupancy of the channel by toxin, is plotted against
kurtoxin concentration (Fig. 5b). The data were well described
by an equation for 1:1 stoichiometry between toxin and chan-
nel with an equilibrium dissociation constant (Kd) of 15 nM.
We conclude that kurtoxin binds to the α1G T-type calcium
channel with high affinity and that the stoichiometry between
toxin and channel is probably 1:1.
We examined the selectivity of kurtoxin for different types of
voltage-gated calcium channels. At 350 nM, kurtoxin almost
completely inhibited the α1G T-type calcium channel and inhib-
ited the α1H T-type calcium channel22 (closely related to α1G)
by about 85% (Fig. 6). This suggests that kurtoxin binds to α1H
with nearly as high an affinity as α 1G (Fig. 5 and legend to

Fig. 4. Kurtoxin slows activation and inactivation of α1G T-type cal-

τ inactivation (ms)

cium channels. (a) Currents elicited by 50-ms depolarizations to

various voltages in the absence (open circles) or presence (closed
circles; 350 nM) of kurtoxin. Holding voltage was –90 mV, and tail
voltage was –60 mV. Dashed lines indicate zero current. Linear
capacitive current and very small background conductances have
been subtracted using 1 mM CdCl2. (b) Scaled currents elicited by
depolarization to either 0 mV or +100 mV in both the absence and
presence of 350 nM kurtoxin. (c) τ inactivation plotted as a function
10 ms
Test voltage (mV) of test voltage in control and 350 nM kurtoxin.

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 © 1998 Nature America Inc. • http://neurosci.nature.com
a b
I
—
Io ρ0
Test voltage (mV)
Fig. 5. Concentration dependence of inhibition by kurtoxin. (a) Fraction of uninhibited current
for various strength depolarizations in different concentrations of kurtoxin. Holding voltage was
–90 mV. I is peak inward current in the specified concentration of toxin, and I0 is peak inward and suggests that the toxin recognizes
current in control. (b) Probability unbound (ρ0) plotted against kurtoxin concentration. ρ0 is a structural motif common to both
equal to the fraction of uninhibited current in the plateau phase at negative voltages, typically channels. Similar cases of toxin
near –50 mV. Data points are mean ± standard error for 3–6 determinations. Smooth curve is a promiscuity have been reported for
fit of ρ0 = (1 + ([kurtoxin]/Kd)) to the data.
–1
© 1998 Nature America Inc. • http://neurosci.nature.com
Fig. 6). In contrast, the same concentration of kurtoxin did not
affect α1B (the N-type calcium channel) 23, α1A (the P/Q-type
calcium channel)24, α1C (an L-type calcium channel)25 or α1E
(ref. 26), suggesting that the Kd of kurtoxin binding to these
channels is probably greater than 10 µM. Thus, kurtoxin dis-
tinguishes between α1G and the high-voltage-activated calcium
channels with over 600-fold selectivity.
The sequence homology between kur-
toxin and the other sodium channel tox-
P 350 nM kurtoxin
ins ranges from 30 to 60% (Fig. 7a), α1G
which raised the possibility that kurtoxin
also interacts with voltage-gated sodium
channels. Therefore, we tested whether
kurtoxin influences the rat brain IIA
sodium channel α subunit coexpressed
27
with rat β1 (ref. 28). Application of kur-
toxin produced a pronounced slowing of
both activation and inactivation kinetics
(Fig. 7). These results indicate that kur-
toxin binds to the sodium channel and
modifies channel gating. The effect of the
toxin on inactivation is similar to what α1B
has been observed for other α-scorpion (+ β1B, α2δ)
toxins18,29–32.
Discussion
Protein toxins that bind to ion channels
have been invaluable tools for studying
the structural basis of channel function
and for investigating the physiological
roles of particular channel subtypes. By
screening for ligands that bind to the α1G
T-type calcium channel, we identified
kurtoxin, a 63 amino-acid protein toxin α1E
found in venom of the Parabuthus trans- (+ β1B, α2δ)
vaalicus scorpion. Kurtoxin binds with
high-affinity (Kd = 15 nM) to what seems
to be a single site on the α1G T-type cal-
cium channel and inhibits the channel in
a voltage-dependent manner. The simi-
larities between the effects of kurtoxin on
the T-type calcium channel and the
nature neuroscience • volume 1 no 8 • december 1998
articles
effects of known gating modi-
fiers 19–21 on other voltage-gated
channels argue that kurtoxin inhibits
the T-type calcium channel by mod-
ifying channel gating. This conclu-
sion is strengthened by the sequence
homology between kurtoxin and the
α-scorpion toxins, a group of well
studied toxins that modify the gating
of sodium channels18.
[kurtoxin] (M) The cross-reactivity of kurtoxin
with the α1G T-type calcium channel
and a sodium channel is remarkable
33
grammotoxin (a calcium channel
inhibitor)19 and hanatoxin (a potas-
sium channel inhibitor)21, two relat-
ed toxins from tarantula venom. Each
of these toxins exhibits cross-reactivity with voltage-gated cal-
cium and potassium channels and inhibits these channels by
modifying the energetics of channel gating33. At least some of
the same residues, for example, Glu 277 and Phe 273 in the
S3–S4 loop of the drk1 potassium channel, contribute to form
receptors for both hanatoxin and grammotoxin33,34. We spec-
p control
Fig. 6. Selectivity of kurtoxin for differ-
ent voltage-gated calcium channels.
α1H Superimposed traces were recorded in
control or 350 nM kurtoxin. Holding
voltage was –90 mV, and depolarizations
were to –40 mV for α1G and α1H, +20
mV for α1B, –5 mV for α1A, +10 mV for
α1E and –10 mV for α1C. Dashed lines
indicate zero current. All high-voltage-
activated calcium channel α subunits
were expressed with β1b and α2δ. Charge
carrier was 5 mM barium. Linear capaci-
tive current and very small background
conductances (< 10 nA from –90 to +50
mV) were isolated by blocking calcium-
α1A
(+ β1B, α2δ)
channel current with CdCl2 and have
been subtracted. 100 µM CdCl2 was used
for blocking all high-voltage-activated
channels, whereas 1 mM CdCl2 was used
for blocking α1G and α1H. Mean ± stan-
dard error (n = 3) values for ratio of cur-
rent in 350 nM kurtoxin divided by
current in control (at voltages that elicit
peak inward current) for the high-volt-
age-activated channels were as follows:
α1B, 0.99 ± 0.036; α1A, 1.00 ± 0.015; α1E,
0.97 ± 0.021; α1C, 1.03 ± 0.035. Mean ±
α1C
standard error (n = 5–6) for ratio of cur-
(+ β1B, α2δ)
rent in 350 nM kurtoxin divided by cur-
rent in control (both elicited by
depolarization to –40 mV) for the low-
voltage-activated channels were as fol-
lows: α1H, 0.15 ± 0.011; α1G, 0.035 ±
0.0053. A value of 0.15 for the fraction of
uninhibited current (at 350 nM kurtoxin )
for α1H corresponds to a Kd of 61 nM.
671
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articles

Fig. 7. Kurtoxin slows inactiva-

tion of voltage-gated sodium
a
channels. (a) Amino-acid
sequence of kurtoxin (Ktx)
compared to eleven other α-
scorpion toxins. Shading of
residues in all toxins (except
kurtoxin) indicates conservation
with kurtoxin. Shading in kur-
toxin indicates the residue is
conserved in at least one toxin
below. (b) Effect of kurtoxin on
a voltage-gated sodium channel.
The rat brain IIA α subunit and
rat β1 subunit were coex- b
pressed. (left traces) Currents
elicited by depolarization to –5
mV in the presence or absence
of 500 nM kurtoxin. Holding
© 1998 Nature America Inc. • http://neurosci.nature.com

voltage was –90 mV. Multiple

traces showing currents elicited
by various strengths of depolar-
ization in control (middle) or
500 nM kurtoxin (right).
10 ms
Depolarizations were to volt-
ages between –60 mV and +10
mV in 5-mV increments. Dashed lines indicate zero current. Linear capacitive current and very small background conductances (<10
nA), isolated by blocking sodium channels with 1 µM tetrodotoxin, have been subtracted.

ulate that kurtoxin binds to a region of the cloned T-type cal-
cium channels and the sodium channel that is homologous to
the hanatoxin/grammotoxin receptor on voltage-gated potas-
sium and calcium channels. Glu 1613 in the S3–S4 loop of
repeat IV in the rat brain IIA sodium channel is homologous to
Glu 277 in the potassium channel33, and mutation of Glu 1613
has a large effect on the binding affinity of an α-scorpion
toxin32. Kurtoxin is an α-scorpion toxin, defined by its amino-
acid sequence and by its ability to bind to sodium channels and
slow inactivation (Fig. 7). Moreover, its effect on the α1G T-
type calcium channel (Figs 3 and 5) is similar to the effects of
hanatoxin and grammotoxin on both voltage-gated potassium
and calcium channels19,21,33. It is interesting that only in repeat
IV of α 1G and α 1H T-type calcium channels does the S3–S4
loop have a Glu at the position equivalent to that of Glu277 in
the drk1 potassium channel and Glu1613 in the sodium chan-
nel. Perhaps only the voltage-sensing domain in repeat IV of
T-type calcium channels contains a competent kurtoxin bind-
ing site. This would explain why our experiments indicate a
1:1 stoichiometry between toxin and channel, even though
there are four homologous voltage-sensing domains in T-type
calcium channels. Two general implications arise from these
comparisons. The first is that many gating-modifier toxins
interact with a very similar region of the voltage-sensing
domains of voltage-gated ion channels. A Glu residue at the C
terminal edge of S3 (in the S3–S4 loop) seems to be important
for the binding of hanatoxin, grammotoxin and the α-scorpi-
on toxins. The second implication is that the structure of these
domains, as seen by the gating modifier toxins, has been high-
ly conserved between members of the voltage-gated ion chan-
nel superfamily.
The subunit composition of native T-type calcium chan-
nels remains to be determined and has become the subject of
some controversy. The α1G and α1H subunits each form calci-
um channels that are similar to T-type calcium channels in
many native cells16,22. However, other types of voltage-gated
calcium-channel α subunits have been implicated as giving rise
to low-voltage-activated calcium channels 35–38 . Kurtoxin
should be a useful reagent to help clarify these issues because it
nicely distinguishes between α1G, α1H and the other voltage-
gated calcium-channel α subunits. In general, kurtoxin should
be a valuable tool for defining both the subunit composition
of native T-type calcium channels and the involvement of T-
type calcium channels in electrical and biochemical signaling.
Methods
E XPRESSION OF VOLTAGE - GATED CALCIUM AND SODIUM CHANNELS IN
X ENOPUS OOCYTES . To improve expression of the rat α 1G calcium
channel16, its cDNA was subcloned into the pGEM-HEA vector, thus
adding the 5’ and 3’ untranslated regions of a Xenopus β-globin gene.
pGEM-HEA was initially created from pGEM-HE 17 by introducing a
unique AflII restriction site downstream from the 3’ untranslated
region of the Xenopus β-globin gene using PCR, verified by DNA
sequencing. α1G was excised from pSP72 by EcoRV digestion. pGEM-
HEA was digested with SmaI and HindIII, ends filled in using Klenow
and blunt-end ligated with the EcoRV fragment of α1G. Correct ori-
entation was initially determined by SacI restriction mapping and
then verified by DNA sequencing around restriction sites. The α1G
-pGEM-HEA construct was linearized with AflII. The human α 1H
cDNA 22 was also subcloned into pGEM-HEA by excising it from
pcDNA3 (Invitrogen, Carlsbad, California) by digestion with EcoRV
and BspEI, then BspEI and XbaI. Both fragments were ligated into
SmaI- and XbaI-digested pGEM-HEA. The α 1H-pGEM-HEA con-
struct was linearized with BfrI. To express the rat brain α1B calcium
channel23 in oocytes, its cDNA was also subcloned into pGEM-HEA.
α 1B was excised from pRC/CMV (provided by T. Snutch) by ClaI
digestion, ends filled in using Klenow, and blunt-end ligated with
the Klenow-treated, SmaI/HindIII-digested pGEM-HEA described
above. Correct orientation was initially determined by restriction
mapping using StyI and then verified by DNA sequencing around

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 © 1998 Nature America Inc. • http://neurosci.nature.com
restriction sites. The α1B -pGEM-HEA construct was linearized with
AflII. Human α1E (pHBE239)26 was linearized with HindIII. Rabbit
cardiac α1C in the pAGA2 vector containing an N-terminal deletion
(DN60)25 was linearized with HindIII. cDNAs encoding rabbit brain
α1A (BI-1; ref. 24), rat brain β1b (ref. 39) and rabbit skeletal muscle
α2δ40 were provided by L. Birnbaumer in the pAGA2 vector41. α1A
and α2δ were linearized with XhoI; β1b was linearized with HindIII.
The rat brain IIA sodium channel α subunit27 and the rat β1 sub-
unit28, both in bluescript vectors (provided by D. Hanck and J. Kyle),
were linearized with XhoI and BamHI, respectively. All transcripts
of calcium and sodium channel subunits were synthesized using T7
RNA polymerase. α1G (~1–10 ng per oocyte) and α1H (~100–500 pg
per oocyte) cRNAs were injected alone into oocytes 4 to 9 days before
recording. α1A, α1B, α1C and α1E cRNAs (~100 pg to 1 ng per oocyte)
were injected with approximately equal quantities of β 1b and α 2δ
cRNAs 24 to 48 h before recording. The rat brain IIA α subunit and
β1 cRNAs were co-injected at equal concentrations (~100 pg per
oocyte) 24–48 h before recording.
ELECTROPHYSIOLOGY. Oocytes from Xenopus laevis frogs were removed
surgically and incubated with agitation for 1.5 h in a solution con-
© 1998 Nature America Inc. • http://neurosci.nature.com
taining NaCl (82.5 mM), KCl (2.5 mM), MgCl 2 (1 mM), HEPES
(5 mM) and collagenase (2 mg/ml; Worthington Biochemical Corp.,
Freehold, New Jersey), pH 7.6 with NaOH. Defolliculated oocytes
were injected with cRNA and incubated at 17°C in a solution con-
taining NaCl (96 mM), KCl (2 mM), MgCl2 (1 mM), CaCl2 (1.8 mM),
HEPES (5 mM) and gentamicin (50 µg/ml; Gibco BRL, Gaithersburg,
Maryland), pH 7.6 with NaOH. Oocyte membrane voltage was con-
trolled using an OC-725C oocyte clamp (Warner Instruments). Data
were filtered at 1–2 kHz (8-pole Bessel) and digitized at 10-20 kHz.
Microelectrode resistances were 0.1 to 0.8 MΩ when filled with 3 M
KCl. Oocytes were studied in a 200 µl recording chamber perfused
with one of two different extracellular solutions. The extracellular
solution for recording of calcium channel currents contained BaOH 2
(5 mM), NaCH3SO 3 (100 mM) and HEPES (10 mM), pH 7.6 with
NaOH. Contamination of calcium-activated chloride current was
minimized by recording in chloride-free solution and recording bar-
ium currents usually less than 2 µA (mostly ~ 1 µA). Sodium chan-
nel currents were recorded in an extracellular solution containing
NaCl (100 mM), MgCl 2 (1 mM), CaCl 2 (0.3 mM) and HEPES
(10 mM), pH 7.6 with NaOH. Cytochrome C (1 mg/ml) was includ-
ed in the extracellular recording solution for experiments examining
the concentration dependence of toxin inhibition (Fig. 5). Agar salt
bridges containing 1 M NaCl were used to connect the ground elec-
trode pools and the recording chamber. All experiments were done
at room temperature (~22–25°C).
TOXIN PURIFICATION AND BIOCHEMICAL CHARACTERIZATION. Lyophilized
Parabuthus transvaalicus venom (Latoxan, France) was dissolved in
25% aqueous acetonitrile with 0.1% trifluoroacetic acid (TFA) at a
concentration of 5 mg/ml. The resulting solution was filtered (0.2 µm,
Gelman, Acrodisc), concentrated under vacuum and fractionated in
two steps using a Beckman reversed-phase HPLC. The first separation
employed a 1 × 25cm C-18 (5 µ, 90 Å) ultrasphere column (Beckman
Inst., Columbia, Maryland) eluted with a linear gradient from 25%
mobile phase B to 100% mobile phase B over 60 min at a flow rate of
3 ml/min, where mobile phase A was 0.1% TFA in H20 and mobile
phase B was 0.08% TFA in acetonitrile. The fraction with highest activ-
ity was collected at about 21 min (Fig. 1a). This active fraction was
subsequently fractionated using a 0.46 × 25cm C-18 (5 µ, 90 Å) ultra-
sphere column (Beckman Inst., Columbia, Maryland) eluted with a
linear gradient from 40% mobile phase B to 100% mobile phase B over
60 min at a flow rate of 0.9 ml/min, where mobile phase A was 0.1%
TFA in H 20 and mobile phase B was 0.08% TFA in methanol. The
inhibitory activity eluting at about 22 min (Fig. 1b) seemed to coin-
cide with a symmetrical peak after re-chromatography on the same
column (Fig. 1c) and was therefore chemically analyzed further. The
mass of the inhibitor was determined on a described42 microbore LC-
electrospray ionization mass spectrometry system to be 7386.1 dal-
tons (average of [M+4H]4+ ion at m/z 1847.3, [M+5H]5+ ion at m/z
1478.3, most intense ion, and [M+6H] 6+ ion at m/z 1232.1). Before
nature neuroscience • volume 1 no 8 • december 1998
articles
amino-acid sequencing, the inhibitor was reduced and alkylated.
Approximately 1 nmole of the inhibitor was reduced in 15 mM dithio-
threitol in 0.4 M ammonium bicarbonate/8 M urea for 30 min at 50ºC.
The sample was allowed to cool to room temperature and alkylated in
15 mM iodoacetamide for 30 min. The reduced and alkylated inhibitor
was purified by reversed-phase HPLC and subjected to automated
Edman degradation on a described system43 resulting in the unequiv-
ocal determination of the identity of the first 62 residues, except for
some uncertainty in the assignment of residues 51 and 57. The reduced
and alkylated inhibitor was digested with chymotrypsin and endo-
proteinase Asp-N essentially according to the method of ref. 44 and
the digests separated by reversed-HPLC as described43. Automated
Edman degradation of the overlapping chymotryptic peptide,
CQGLPDNAR (residues 46–54) and the overlapping C-terminal endo-
proteinase Asp-N peptide DNARIKRSGRCRA (residues 51–63)
resolved the uncertainty in residues 51 and 57 and identified the C-
terminus of the inhibitor. The calculated average molecular weight of
intact inhibitor, 7386.5 daltons, agrees with the experimentally deter-
mined molecular weight of 7386.1. An extinction coefficient (280 nm)
of 2.3 x 104 per M per cm was determined by quantitative amino-acid
analysis and used to measure toxin concentration.
Acknowledgements
We thank Zhe Lu, Chinfei Chen and Yingying Li-Smerin for discussions.
RECEIVED 28 JULY: ACCEPTED 16 O CTOBER 1998
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