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Inhibition of Sodium Channels
in Rat Dorsal Root Ganglion Neurons by Hainantoxin-IV, a Novel Spider Toxin
XIAO Yu-Cheng, LIANG
Song-Ping*
( College
of Life Sciences, Hunan Normal University, Changsha 410081, China )
Abstract The
effects of Hainantoxin-IV (HNTX-IV), a neurotoxic peptide isolated from the
venom of the Chinese bird spider Seleconosmia hainana, on the adult rat dorsal
root ganglion (DRG) neurons were investigated. Using the whole-cell patch-clamp
technique HNTX-IV inhibited mammal neural TTX-sensitive (TTX-S) sodium currents
evidently but the toxin failed to affect TTX-resistant (TTX-R) ones. The
inhibition of HNTX-IV is dose-dependent with the IC50 value of 44.6 nmol/L. The
toxin didn’t affect the activation and inactivation kinetics of sodium
currents, but it caused a 10.1 mV hyperpolarizing shift in the voltage midpoint
of steady-state sodium channel inactivation on DRG neurons. The results
indicated that HNTX-IV, a novel spider toxin, maybe alternate voltage-gated
sodium channels through a mechanism distinct from other spider toxins such as δ-ACTXs,
μ-agatoxins I-VI which targeted the receptor site 3 to slow the inactivation
kinetics of sodium currents.
Key words spider toxin; dorsal
ganglion neurons; sodium current; whole-cell patch-clamp
The venoms of
many animals such as snake, scorpion, marine snail and spider contain many different
kinds of toxins which can affect sodium channels with different mechanism.
These natural toxins, especially tetrodotoxin (TTX) and scorpion α, β-toxins,
are important useful tools for studying the structure-function relationship of
sodium channels. At least six sites (1 – 6) on neural sodium channels have been
disclosed by using these toxins[1]. About twenty spider toxins are
found to alternate sodium channels, such as δ-ACTXs[2], μ-agatoxins
I-VI[3], PhTx2[4]. These spider toxins are peptides consist
of 40-80 residues with four disulfide bonds. They can slow the inactivation
kinetics of currents in a similar manner to α-scorpion toxin by banding the
site 3 on sodium channels[2]. Hainantoxin-IV (HNTX-IV) is a spider
toxin isolated from the venom of the Chinese bird spider Seleconosmia hainana[5].
The sequence of HNTX-IV have been determined to be:
NH2-ECLGFGKGCNPSNDQCCKSSNLVCSRKHRWCKYEI-CONH2 with three disulfide bonds. Its
chemical synthesis has been achieved by a solid-phase method[6]. The
intraperitoneal LD50 values of the toxin in mice is 0.2 mg/kg. It can block
neuromuscular transmission in the isolated nerve-synapse preparations of rat
vas deferens and mouse phrenic nerve-diaphragm. Here we report the effects of
HNTX-IV on voltage-gated sodium currents in adult rat dorsal root ganglion
(DRG) cells.
1 Materials
and Methods
1.1 Purification
of toxin
HNTX-IV was
purified using reverse phase HPLC followed by ion-exchange chromatograph as
described earlier[5].
1.2 Cell
isolation procedures
Rat DRG neurons
were acutely dissociated and maintained in a short-term primary culture using
the method described by Wang et al.[7]. Briefly, 30-day adult
Sprague-Dawley rats of either sex were killed by decapitation and the dorsal
root ganglia were removed quickly from the spinal cord, and then they were
transferred into Dulbecco’s modified Eagle’s medium (DMEM) containing trypsin
(0.5 g/L, type III, Sigma), collagenase (1.0 g/L, type IA, Sigma) and DNase
(0.1 g/L, type III, Sigma) to incubate at 34 ℃ for 30 min. Trypsin inhibitor (1.5 g/L, type II-S, Sigma) was used
to terminate enzyme treatment. After transferred into 35 mm culture dishes
(corning, Sigma), the DRG cells were incubated in CO2 incubator (5%
CO2, 95% air, 37 ℃) for 1 – 4 h before patch-clamp experiment.
1.3 Electrophysiological
recordings
Sodium currents
(filtered at 10 kHz, digitized at 3 kHz with a EPC-9 patch-clamp amplifier,
HEKA Electronics, Germany) were recorded at room temperature (20 – 25 ℃). Micropipettes (2 – 3 μm
diameter) were pulled from borosilicate glass capillary tubing by using a
two-step vertical puller (PC-10, Narishige, Olypmus) and heat-polished with a
microforge (MF-900, Narishige). The resistances of micropipettes were 1 – 2 MΩ after filled with internal
solution contained (mmol/L): CsF 135, NaCl 10, HEPES 5, with pH adjusted to 7.0
with 1 mol/L CsOH. The external bathing solution contained (mmol/L): NaCl 30,
CsCl 5, D-glucose 25, MgCl2 1, CaCl2 1.8, HEPES 5,
tetraethylammonium (TEA) chloride 20, tetramethylammonium chloride 70, with pH adjusted
to 7.40 with 1 mol/L TEA hydroxide[2]. The osmolarities of both
internal solution and external solution were adjusted to 290 mOsmol with
sucrose. An Ag-AgCl pipette/150 mmol/L NaCl-agar bridge was introduced between
bath electrode and bathing solution to avoid disturbing the composition of the
external solution. After establishing the whole-cell recording configuration,
experiments didn’t commence for a period of more than 3 – 4 min to allow
adequate equilibration between the micropipette solution and the cell interior.
Drug-containing solutions of about 10 μL volume were applied by pressure
injection with a microinjector (IM-5B, Narishige). All chemical reagents were
purchased from Sigma.
2 Results
2.1 Effects
of HNTX-IV on voltage-gated sodium currents
The DRG cells
with diameters of 20 – 40 μm were selected for experiments, for larger DRG
cells from older animals tend to express fast TTX-S sodium currents while
smaller ones (10 – 20 μm) tended to express slow TTX-resistant (TTX-R) sodium
currents[8]. TTX (200 nmol/L) was added into external solution
to separate TTX-R sodium currents from TTX-S ones. Under voltage-clamp conditions,
sodium currents on DRG cells were elicited by 50 ms depolarization to -10
mV from a holding potential of -80 mV every 1 min. Fig. 1(A) showed that HNTX-IV
reduced the peak amplitude of TTX-S sodium currents to a maximum effect within
3 min on DRG neurons.10 and 50 nmol/L HNTX-IV could reduce control currents
amplitude by (28.8±6.5)% and (57.2±5.4)% (
±s), respectively. The reductions of HNTX-IV
were concentration-dependent with the IC50 value of 44.6 nmol/L [Fig.1(C)].
After reduction, the shape of sodium currents was similar to that of control,
indicating that HNTX-IV didn’t change the activation and inactivation kinetics
of TTX-S sodium currents. In contrary, TTX-R sodium currents weren’t inhibited
significant on DRG cells by 50 nmol/L HNTX-IV [Fig. 1(B)], implying that HNTX-IV
didn’t alternate the steady-state activation and inactivation kinetics of
TTX-R sodium channels.
Fig.1 Effects
of HNTX-IV on the TTX-S(A) and TTX-R sodium currents(B) and the dose-dependent
block of TTX-S sodium currents(C)
Currents were induced by a 50 ms depolarizing
potential of –10 mV from a holding potential of –80 mV. In (C) data points
(
±s) were fitted according to Boltzmann equation.
2.2 Effects
of HNTX-IV on the current-voltage relationship of TTX-sensitive sodium channel
The
current-voltage curve of TTX-S sodium channels were obtained by depolarization
steps from a holding potential of -80 mV to +50 mV. Under control conditions
TTX-S sodium currents were initial elicited at the –50 mV and reached maximal
amplitude at around -20 mV. After 50 nmol/L HNTX-IV treatment for 3 min, both
the threshold of activation and the active voltage of peak inward currents
weren’t changed, and the membrane reverse potential wasn’t shifted evidently,
too (Fig. 2).
Fig.2 Effects of
HNTX-IV on rat DRG peak sodium currents (n=5)
(A) Currents were evoked before (a) and
after (b) application of HNTX-IV (50 nmol/L) by 50 ms depolarizing steps from –80
mV to +50 mV in +10 mV increments from the holding potential of –80 mV. (B) The
relationship of voltage and sodium currents in the presence and absent of
HNTX-IV. ●, control; ○, 50 nmol/L HNTX-IV.
2.3 Effects
of HNTX-IV on inactivation kinetics of TTX-S sodium channels
Using a standard
two-pulse protocol, we quantified the changes of the voltage dependence of
steady-state sodium channel inactivation produced by HNTX-IV. Under whole-cell
patch clamp condition, DRG cells were held at a prepulse voltage ranged from
-130 mV to –30 mV in +10 mV increments for 1 s. After 0.5 ms interpulse interval
a test pulse to –10 mV for 50 ms was delivered to evoke TTX-sensitive sodium
currents. Peak sodium currents recorded during the test pulse were normalized
to the maximal value and plotted against the conditioning prepulse potential.
Fig.3 showed that 50 nmol/L HNTX-IV caused the half-maximal inactivation potential
of sodium channels to shift approximate 10.1 mV in hyperpolarizating direction
from (–64.0±1.1) mV to (–74.1±1.6) mV (±s, n=4) and the slop factor (k)
was increased by 2.0 mV from (8.8±0.5) mV of control to (10.8±0.8) mV.
Fig.3 Effects
of HNTX-IV on the steady-state inactivation of TTX-S sodium channels in rat DRG
neurons (n=5)
The curves were fitted by the Boltzmann
equation: I/Imax=1/{1+exp[(V-V1/2)/k]}
where V is holding voltage, V1/2 is the voltage at
which I is 0.5 Imax, and k is the slop factor. ●, control; ○, 50 nmol/L HNTX-IV.
3 Discussion
The results of previous
experiment in our laboratory showed that the contractions of mouse diaphragm
induced by direct electrical stimulus weren’t affected by HNTX-IV, and no
visible symptoms were observed after injection of HNTX-IV into cockroaches
(inacimal dose 60 μg/g body weight)[6], which suggests that HNTX-IV
should target selectively the sodium channel isoforms on mammal neuron
membranes. Although there are two kinds of sodium channels (TTX-S and TTX-R) on
neurons, HNTX-IV inhibited the former selectively similar to other spider
toxins such as δ-ACTXs[2]. Nanomolar concentration of the toxin
evidently inhibited sodium currents induced on adult rat DRG neurons. HNTX-IV
didn’t shift the membrane reverse potential of sodium channels on DRG neurons,
implying that it didn’t change the ion selectivity of channels. The most
intriguing finding of the present study, however, was that the spider toxin
didn’t affect the inactivation kinetics of TTX-S sodium currents on DRG
neurons. The similar result was also obtained on NG108-15 cells (unpublished
result). Furthermore, the result was consistent with that of the crude spider
venom which didn’t slow the inactivation kinetics of sodium currents on
NG108-15 cells[9]. To date almost twenty spider toxins are found to
affect sodium channels, but they all have a common mode of action, similar to
that of α-scorpion toxin and sea anemone toxins by banding the site 3 on sodium
channels. They slow down the inactivation kinetics of currents to prolong the
time course of action potential, so that they cause spontaneous contractions of
isolated rat vas deferens smooth muscle[2] and even increase the
isolated diaphragm muscle twitch tension[4]. In contrary, HNTX-IV
didn’t show such properties but blocked neuromuscular transmission in the
isolated nerve-synapse preparations of rat vas deferens and mouse phrenic
nerve-diaphragm in our previous studies[6]. So, HNTX-IV may be a
novel spider toxin which alternates voltage-gated sodium channels through a
mechanism distinct from other spider toxins.
Acknowlegements We
are grateful to Prof. LI Zhi-Wang at Tongji Medical University for technical
assistance in DRG cell isolation.
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