|
http://www.abbs.info e-mail:[email protected] ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(1): 82-85 CN 31-1300/Q |
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.
References
1 Cestele S, Ben Khalifa RB, Pelhate M, Rochat H, Gordon D. α-Scopion toxins binding on rat brain and insect sodium channels reveal divergent allosteric modulations by brevetoxin and veratridine. J Biol Chem, 1995, 270(25): 15153 – 15161
2 Szeto TH, Birinyi-Strachan LC, Smith R, Connor M, Christie MJ, King GF, Nicholson GM. Isolation and pharmacological characterization of δ-atracotoxin-Hv1b, a vertebrate-selective sodium channel toxin. FEBS Lett, 2000, 470(3): 293 – 299
3 Skinner WS, Adams ME, Quistad GB, Kataoka H, Cesarin BJ, Enderlin FE, Schooley DA. Purification and characterization of two classes of neurotoxins from the funnel web spider, Agelenopsis aperta. J Biol Chem, 1989, 264(4): 2150 – 2155
4 Araujo DA, Cordeiro MN, Diniz CR, Beirao PS. Effect of a toxic fraction, PhTx2, from the spider Phoneutria nigriventer on the sodium current. Naunyn Schmiedebergs Arch Pharmacol, 1993, 347(2): 205 – 208
5 Liang SP, Peng XJ, Huang RH, Chen P. Biochemical identification of Selenocosmia hainana sp. nov. from south China [Araneae, Theraphosidae]. Life Science Research, 1999, 3(4): 299 – 303
6 Liu ZH, Chen P, Liang SP. Synthesis and oxidative refolding of Hainantoxin-IV. Acta Biochim Biophys Sin, 2002, 34(4): 516 – 519
7 Wang QW, Li Q, Li ZW. Inhibitory effects of clonidine on GABA-activated currents in DRG neurons. Acta Physiol Sin, 1998, 50(1): 19 – 27
8 Su X, Wachtel RE, Gebhart GF. Capsaicin sensitivity and voltage-gated sodium currents in colon sensory neurons from rat dorsal root ganglia. Am J Physiol, 1999, 277(6 Pt 1): G1180 – G1188
9 Xiao YC, Peng K, Chen XD, Liang SP. Inhibition of voltage-gated sodium channels in NG108-15 by the venoms from three kinds of spider. Life Science Research, 2001, 5(5): 42 – 47