The modulation effects of BmK I, an α-like scorpion neurotoxin, on voltage-gated Na+ currents in rat dorsal root ganglion neurons

Neuroscience Letters 390 (2005) 66–71

The modulation effects of BmK I, an ␣-like scorpion neurotoxin, on voltage-gated Na+ currents in rat dorsal root ganglion neurons Jin Chen b , Zhi-Yong Tan c , Rong Zhao b , Xing-Hua Feng b , Jian Shi b , Yong-Hua Ji a,b,∗ b

a School of Life Sciences, Shanghai University, Shang-Da Road 99, Shanghai 200444, PR China Graduate School of the Chinese Academy of Sciences, Shanghai Institute of Physiology, Institutes for Biological Sciences, Chinese Academy of Sciences, PR China c Department of Anesthesiology, Yale University School of Medicine, New Haven, CT, USA

Received 3 June 2005; received in revised form 21 July 2005; accepted 1 August 2005

Abstract The present study investigated the effects of BmK I, a Na+ channel receptor site 3 modulator purified from the Buthus martensi Karsch (BmK) venom, on the voltage-gated sodium currents in dorsal root ganglion (DRG) neurons. Whole-cell patch-clamping was used to record the tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) components of voltage-gated Na+ currents in small DRG neurons. It was found that the inhibitory effect of BmK I on open-state inactivation of TTX-S Na+ currents was stronger than that of TTX-R Na+ currents. In addition, BmK I exhibited a selective enhancing effect on voltage-dependent activation of TTX-S currents, and an opposite effect on time-dependent activation of TTX-S and TTX-R Na+ currents. The results suggested that the inhibitory effect of BmK I on open-state inactivation might contribute to the increase of peak TTX-S and TTX-R currents, and the enhancing effect of BmK I on time-dependent activation might also contribute to the increase of peak TTX-S currents. It was further suggested that a combined effect of BmK I including inhibiting the inactivation of TTX-S and TTX-R channels, accelerating activation and decreasing the activation threshold of TTX-S channels, might produce a hyperexcitability of small DRG neurons, and thus contribute to the BmK I-induced hyperalgesia. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Sodium channel; Subtype; Scorpion toxin

Sodium channels are the target for modulators of diverse origins and chemical structures. Binding to specific receptor sites, these modulators have effects that range from pore blocking to modification of the gating and permeation (for review see [3]). Scorpion ␣/␣-like toxins are long-chain polypeptides from scorpion venoms that can prolong the action potential on excitable cells by inhibiting the open-state inactivation of the voltage-gated Na+ channels (VGSCs). They bind to the receptor site 3 on VGSCs, which is partially located in the short external loop connecting the S3 and S4 segments of domain 4 [3]. BmK I, a putative sodium channel modulator purified from the venom of Buthus martensi Karsch (BmK) [8], showed typical binding properties as an ∗

Corresponding author. Tel.: +86 21 66135189x54920300. E-mail addresses: [email protected], [email protected] (Y.-H. Ji). 0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.08.003

␣-like scorpion toxin to insect sodium channels with high affinity, but lack of the specific binding on rat brain synaptomesomes [10] and little effect on Nav 1.2 current expressed on Xenopus oocyte [11]. However, subcutaneous (s.c.) administration of BmK I to rat could induce nociceptive response [4], as well as increase c-Fos expression in spinal cord [1]. These results implied that the BmK I might have some specific effect action on the sodium channels expressed in peripheral nerve system of rat. Sensory neurons with their cell bodies in dorsal root ganglia (DRG neurons) transmit various types of sensory information from the periphery to the spinal cord. DRG neurons are a physically and functionally heterogeneous population [13]. To date, several subtypes of ␣-subunit and all four subtypes of ␤-subunit have been detected in rat DRG neurons [2,12,19,20]. Thus, to find the possible targets for BmK I in peripheral nerve system, acutely dissociated DRG neurons

J. Chen et al. / Neuroscience Letters 390 (2005) 66–71

were chosen for whole-cell patch clamping to investigate the possible effects of BmK I on TTX-S and TTX-R currents. Crude venom of the scorpion Buthus martensi Karsch (BmK) was purchased from a private farm in Suzhou, Jiangsu Province, China PR. BmK I was purified according to the methods described by Ji et al. [8]. Sprague–Dawley rats were from the Shanghai experimental animal center. Cells were isolated from the DRG of adult male rats (120 ± 12 g) according to the method described by [17]. Briefly, rats were killed by concussion with subsequent cervical dislocation. Ganglia were dissected from the full length of the vertebral column. The trimmed ganglia were digested with collagenase type II (1.0 mg/ml, Sigma, USA) and trypsin type I (0.5 mg/ml, Sigma, USA) at 37 ± 0.5 ◦ C for about 30 min. Single cells were dissociated mechanically with a series of fire-polished Pasteur pipettes, and then plated into dishes. Whole-cell voltage-clamp experiments were performed as described previously [6], using an EPC-9 amplifier (HEKA eletronik, Germany) at room temperature. Patch pipettes were fabricated from glass capillary tubes by PP-830 Puller (Narishige, Japan) with the resistance of 1–3 M
. Data acquisition and stimulation protocols were controlled by a Pentium III computer (Legend) equipped with Pulse/PusleFit 8.3 software (HEKA eletronik, Germany). Capacitance transients were cancelled and series resistance was compensated (>75%). Leak subtraction was performed using P/4 protocol. Data were low-passed at 10 kHz. The pipette solution was composed of (in mM): CsCl 140, Na2 -ATP 3, HEPES 10, CaCl2 0.5, EGTA 5. The pH of the solution was adjusted to 7.4 with CsOH. The bath solution contained (mM): NaCl 115, TEA-Cl 20, KCl 5, CaCl2 0.01, MgCl2 5, HEPES 10, glucose 5 (pH 7.4 with NaOH). TTX-S and TTX-R INa were distinguished according to the method described by [17]. Briefly, 1 ␮M TTX (Sigma, USA) was used to separate the TTX-R INa from TTX-S INa . After complete inhibition of TTX-S portion using 1 ␮M TTX, the whole-cell INa were recorded in the external solution containing both BmK I and 1 ␮M TTX, and then the cells were washed with the external solution containing 1 ␮M TTX. The fast kinetics of TTX-S channels enabled the TTX-S INa to be distinguished from the TTX-R INa . Neurons expressing INa that completely inactivated at the end of a 10 ms depolarizing pulse to 0 or −10 mV were chosen for the recording of TTX-S INa . Toxin was dissolved in the bath solution, supplemented with 1 mg/ml bovine serum albumin (BSA) in order to prevent adherence of toxin to the vials and the perfusion apparatus. Application of 1 mg/ml bovine serum albumin (BSA) alone did not alter Na+ channel function. Throughout the experiment, bath or toxin solutions were delivered to the cells via a fast gravity-driven perfusion system. The silicon barrel tip for drug application was ∼200 ␮m from the cells. Data were analyzed by PulseFit 8.5 (HEKA eletronik, Germany), Sigma-Plot (Jandel Scientific, San Rafeal, CA, USA) and Origin 7.0 (Northampton, USA). Data were pre-

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sented as mean ± S.E.M. and n represents the number of the cells examined. The time-dependent activation and inactivation were described with the model of Hodgkin and Huxley [7]: I(V, t) = gNa (V − VNa ) × [1 − exp(−t/τ m )]3 exp(−t/τ h ), where gNa was the maximum Na+ conductance of the channels achieved in the absence of inactivation, and τ m and τ h are the time constants of activation and inactivation, respectively. The sodium conductance (gNa ) at a different potential was calculated: gNa = INa /(V − VNa ), where VNa is the reversal potential of INa . The voltage dependence for activation of TTX-R and TTX-S INa was fitted with the Boltzmann relation: g/gmax = 1/[1 + exp(Vg − V)/kg ], where Vg is the voltage for half-maximum activation, and kg is the slope factor. The steady-state inactivation parameter (h) was calculated by dividing the current achieved following a given prepulse by the maximum test pulse current achieved. The prepulse potential is termed Vh at h = 0.5. The slope factor of the h curve is termed kh at the mid-point (h = 0.5). Vh and kh were determined from a least-squares fit to the data of the relationship: h(I/Imax ) = 1/[1 + exp(V − Vh )/kh ]. Statistical effects of BmK I were analyzed by Student’s t-test and the significance level was set to 0.05. The membrane potential was held at −70 mV. The INa was elicited by a series of steps in potential from −60 to +60 mV with a 10 mV increment. Fig. 1 shows the recordings of families of TTX-S and TTX-R INa on DRG neurons in the absence (Fig. 1A(a)) and presence (Fig. 1A(b)) of 200 nM BmK I. The maximal inward current among the current family, which was defined as peak INa , was chosen to evaluate the effect of BmK I on the current amplitude. In control condition, TTX-S peak INa emerged between −10 to 0 mV, while TTX-R peak INa appeared between 0 to 10 mV. The normalized and averaged TTX-S peak INa was dose-dependently increased from control (100%) to 113 ± 2% (n = 7), 120 ± 4% (n = 8), 129 ± 13% (n = 6), and 146 ± 6% (n = 4) after the perfusion of 100, 200, 500 and 1000 nM BmK I, respectively (Fig. 1B). Meanwhile, the TTX-R peak INa was also increased in a dose-dependent manner. The presence of 100, 200, 500 and 1000 nM BmK I caused the peak TTX-R INa increased to 109 ± 2% (n = 7), 112 ± 2% (n = 9), 115 ± 1% (n = 10), and 118 ± 1% (n = 9), respectively (Fig. 1B). Washing for 10 min or more could partially reverse the effect of BmK I on current amplitude. BmK I administration produced the effects on the timedependent activation and inactivation of TTX-S INa , as well as on TTX-R INa . Fig. 2 showed the TTX-S (Fig. 2A(a)) and TTX-R (Fig. 2A(b)) Na+ currents were induced by depolarizing from the holding potential of −70 mV to 0 mV before (solid lines) and after (dashed lines) the treatment of 200 nM BmK I. Both currents were fitted by the m3 h model of Hodgkin and Huxley [7]. The averaged τ m (activation time constant) and τ h (inactivation time constant) during the depolarization were plotted in Fig. 2B as a function of the test potential. BmK I accelerated activation and removed fast inactivation of TTX-S INa (Fig. 2B(a), Table 1). However, both activation and inactivation of TTX-R INa were slowed in

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J. Chen et al. / Neuroscience Letters 390 (2005) 66–71

Fig. 1. The effect of BmK I on TTX-S and TTX-R INa . (A) Representative TTX-S (left panel) or TTX-R (right panel) INa evoked by depolarization ranging between −60 and 60 mV by a 10-mV increment from holding potential of −70 mV, before (a) and after (b) BmK I 200 nM treatment in the same cell. (B) The maximal INa among the current family was defined as peak INa and used to evaluate the effect of BmK I on current amplitude. BmK I application increased TTX-S and TTX-R peak INa in a dose-dependent manner. Treated with 100, 200, 500 and 1000 nM BmK I, the normalized and averaged TTX-S peak INa was increased to 113 ± 2% (n = 7), 120 ± 4% (n = 8), 129 ± 13% (n = 6), 146 ± 6% (n = 4), respectively. On the contrast, the TTX-R INa increased to 109 ± 2% (n = 7), 112 ± 2.5% (n = 9), 115 ± 1% (n = 10), and 118 ± 1.3% (n = 9), respectively (t-test, * P < 0.05; ** P < 0.01 vs. control).

the presence of same concentration of BmK I (Fig. 2B(b), Table 1). The midpoint voltages (V1/2 ) and slope for activation of TTX-S and TTX-R INa were obtained from fitting the conductance–voltage curve with the Boltzmann equation (Fig. 3A and Table 2). BmK I 200 nM negatively

shifted activation of TTX-S INa from −14.71 ± 0.18 mV to −18.53 ± 0.35 mV (Fig. 3A(a), Table 2), while there is no significant effect of BmK I on activation of TTX-R INa with the V1/2 moving from −4.22 ± 0.67 mV to −2.55 ± 0.59 mV at the same concentration (Fig. 3A(b), Table 2). The slope factor variations of TTX-S and TTX-R INa were within ±1 mV

Fig. 2. The effect of BmK I on the time-dependent activation and inactivation of TTX-S and TTX-R INa . (A) TTX-S (a) and TTX-R (b) INa evoked by depolarization from holding potential −70 mV to 0 mV in the absence (solid line) and presence (dashed line) of 200 nM BmK I, respectively. (B) The timedependent activation (τ m , open symbols) and inactivation (τ h , filled symbols) of TTX-S (a) and TTX-R (b) INa were fitted with Hodgkin–Huxley model and plotted as a function of test potential under the control and BmK I-treated conditions. Paired t-test was used for statistical analysis of the difference between control and BmK I treatment (see Table 1).

J. Chen et al. / Neuroscience Letters 390 (2005) 66–71

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Table 1 Statistical comparison of time-dependent activation and inactivation of TTX-S and TTX-R INa TTX-S BmK I (nM) Tau m

Tau h

100 200 500 1000 100 200 500 1000

TTX-R −20

−10

0

10

** *

* ** ** * * ** ** *

** *

*

* * ** *

** *

* * ** *

* ** ** *

20

** * * ** *

30

n

* *

8 5 11 4

* * ** *

8 5 11 4

0

* *

10

30

40

* *

** *

***

***

***

* * * ***

7 9 6 9

* * * **

* ** * *

* ** * *

* * ** *

7 9 6 9

The paired t-test was used to determine the statistically significant difference of τ m and τ h between BmK I-treatment and control *** P < 0.001; BmK I treated vs. control).

before and after BmK I treatment, and did not reach statistical significance (Table 2). The voltage dependence of inactivation of TTX-S and TTX-R INa was determined by eliciting 500-ms conditioning pulses to voltages between −120 and 20 mV in 10-mV increments followed by a standard test pulse to −10 mV. The normalized INa during the depolarization is plotted in Fig. 3B as a function of the prepulse voltage. Data were fit with a Boltzmann function and the half-inactivation voltage

50

n

20

*

(* P < 0.05; ** P < 0.01;

(Vh ) and the slope factors (kh ) were calculated. There is no significant effect of 200 nM BmK I on the steady state inactivation of TTX-S (−71.9 ± 1.1 mV, −7.3 ± 1.0 mV/efold versus −72.1 ± 0.6 mV, −7.4 ± 0.6 mV/e-fold) and TTX-R (−32.4 ± 0.5 mV, 6.1 ± 0.4 mV/e-fold versus −33.0 ± 1.5 mV, 6.20 ± 1.3 mV/e-fold) currents (Fig. 3B, Table 2). The results showed that BmK I caused dose-dependent increase of current amplitude and prolonged of inactivat-

Fig. 3. The effects of BmK I on the voltage-dependent activation and inactivation of TTX-S and TTX-R INa . (A) The voltage dependence of activation of TTX-S (a) and TTX-R INa (b) before (䊉) and after () 200 nM BmK I treatment. Sodium currents were elicited by depolarizing pulses from a holding potential of −70 mV to potentials ranging from −60 to +60 mV in 10 mV increments. Conductance values were calculated by dividing the peak current amplitude by the driving force at each potential and normalizing to the maximum conductance. (B) The voltage dependence of inactivation of TTX-S INa (a) was determined using a two-step protocol in which a 500 ms conditioning pulse to potentials ranging from −120 to 0 mV was followed by a 50 ms test pulse to 0 mV to measure peak current amplitude before (䊉) and after () 200 nM BmK I treatment. The inactivation curve of TTX-R (b) INa was obtained also by the two-step protocol with the condition pulse ranging from −80 to 20 mV followed by a test pulse to 20 mV. Values represent averages, and error bars indicate S.E.M. The data were fit with Boltzmann equation and the parameters of the fits and sample sizes are shown in Table 2.

J. Chen et al. / Neuroscience Letters 390 (2005) 66–71

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Table 2 The voltage dependence of activation and inactivation of TTX-R and TTX-S INa in the absence and presence of BmK I Activation

Inactivation

Vg (mV)

kg (mV/e-fold)

n

Vh (mV)

kh (mV/e-fold)

n

TTX-S Control BmK I

−14.71 ± 0.18 −18.53 ± 0.35 (P < 0.01)

4.82 ± 0.14 5.74 ± 0.32 (P > 0.05)

7 7

−71.85 ± 1.12 −72.07 ± 0.64 (P > 0.05)

−7.27 ± 01.01 −7.44 ± 0.58 (P > 0.05)

7 7

TTX-R Control BmK I

−4.22 ± 0.67 −2.55 ± 0.59 (P > 0.05)

7.66 ± 0.60 7.42 ± 0.53 (P > 0.05)

8 8

−32.41 ± 0.49 −33.03 ± 1.51 (P > 0.05)

−6.14 ± 0.43 −6.20 ± 1.32 (P > 0.05)

6 6

The results are expressed as mean ± S.E.M. of the percentage of BmK I 200 nM. Statistical comparison between the effect of BmK I vs. control was made using paired Student t-test.

ing time constant of both TTX-S and TTX-R currents. The slowed inactivating time constant might contribute to the increase of the amplitude of both TTX-S and TTX-R currents. Thus the stronger effect of BmK I on the amplitude of TTXS currents might be attributed to the stronger effect of BmK I on the inactivation of TTX-S currents, partially. Another effect of the increase of activating time constant would also contribute to the increase of the amplitude of TTX-S currents. DRG neurons co-express multiple Na+ channel mRNAs, correlated with the heterogeneity of INa , including TTX-S and TTX-R INa . In DRG neurons, the TTX-S sodium channels include Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.6, and Nav 1.7. Our previous study demonstrated that BmK I lacked specific binding to the rat synaptomsomes [10] which contained at least Nav 1.2 and Nav 1.1, suggesting BmK I probably targeted neither of them. The later results from expressed Nav 1.2 proved our conjecture [11]. Nav 1.3 shares high homology with Nav 1.1 and Nav 1.2, especially in the extracellular region where scorpion toxins are supposed to bind to [9,15]. Thus, it may also exclude Nav 1.3 as the target for BmK I. Furthermore, the abundance of Nav 1.2 and Nav 1.3 in normal adult DRG neurons is relatively low and the Nav 1.1 expression is low in small diameter DRG neurons. Therefore, the strong effect of BmK I on TTX-S INa may be ascribed to its possible modulation on Nav 1.6 and/or Nav 1.7. It is interesting that BmK I still produces moderate effect on TTX-R INa other than the completely adiaphorous action of those classic ␣ toxins, such as LqTx [16]. Besides Nav 1.8 and Nav 1.9, TTX-R sodium channels on DRG neurons can include low levels of Nav 1.5 [14] which is sensitive to BmK I [5,10,18]. This might partially explain the observable effect on TTX-R INa . Previous studies have not demonstrated whether ␣-like toxins could directly affect Nav 1.8 and/or Nav 1.9. Our results show that BmK I could probably affect the two subtypes of sodium channels expressed in DRG neurons. However, it was worth noting that BmK I showed different potency on various sodium channel isoforms, which could endow it with the ability to identify different isoforms at the level of macroscopic current. The modulation of BmK I on the TTX-S and TTX-R channels in DRG neurons might affect the excitability of nociceptive afferent fiber. First, a BmK I-induced delay of inactivation of TTX-S and TTX-R channels, combined

with a BmK I-induced acceleration of activation of TTX-S channels, might cause a broadened action potential (AP) generated at the AP initiated zone located in the peripheral terminal, and thus cause an increased firing frequency in the nociceptive afferent fiber. Second, a BmK I-induced negative shift of TTX-S activation might decrease the threshold of AP generated at the AP initiated zone. Therefore, the modulation of BmK I on TTX-S and TTX-R channels in DRG neurons might produce hyperexcitability of primary nociceptive afference, and thus contribute to the BmK I-induced hyperalgesia.

Acknowledgements This study was supported by National Basic Research Program of China (2006CB500801), partially by a grant from National Nature Sciences Foundation of China (30370446).

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