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Modulation of BmK AS, a scorpion neurotoxic polypeptide, on voltage-gated Na+ channels in B104 neuronal cell line
Neuroscience Letters 340 (2003) 123–126 www.elsevier.com/locate/neulet
Modulation of BmK AS, a scorpion neurotoxic polypeptide, on voltagegated Naþ channels in B104 neuronal cell line Zhi-Yong Tan, Jin Chen, Hai-Ying Shun, Xing-Hua Feng, Yong-Hua Ji* The Key Laboratory of Neuroscience, Institute of Physiology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China Received 11 December 2002; received in revised form 6 January 2003; accepted 10 January 2003
Abstract The modulation of BmK AS, a neurotoxic polypeptide from scorpion Buthus martensi Karsch, on the voltage-gated Naþ channels has been investigated in the B104 neuroblastoma cell line by whole-cell patch clamping. It was found that the whole Naþ currents and tetrodotoxinresistant Naþ current were depressed in a biphasic manner and both steady-state activation and inactivation curves were shifted toward the hyperpolarizing direction in the presence of BmK AS. The results suggested that BmK AS could inhibit both tetrodotoxin-sensitive and resistant Naþ currents via modulating the gating mechanism of the voltage-gated Naþ channels in B104 cells. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Scorpion neurotoxin; BmK AS; B104; Voltage-gated Naþ channel
Most long-chain scorpion neurotoxic polypeptides, consisting of 60 –70 amino acid residues, are modulators of the voltage-gated Naþ channel (VGSC) in mammals, insects and other vertebrates [11]. According to biological specificity in vivo and pharmacological and electrophysiological activity, these scorpion neurotoxins can be divided into mammal-selective a or b toxins and excitatory or depressant insect-selective toxins [13]. The Asian scorpion Buthus martensi Karsch (BmK) is a species widely distributed in northwestern China, Mongolia and Korea. Up to now, at least ten long-chain neurotoxic components have been purified from BmK venom and determined to be modulators of Naþ channels [5 –7,9,14,15,17]. BmK AS is a unique long-chain polypeptide purified from the venom of B. martensi Karsch. It was found that BmK AS could enhance [3H]ryanodine binding to skeletaltype ryanodine receptors through an indirect mechanism [8], and bind to a distinct receptor site of VGSC on mammal and insect excitable cell membranes in a manner similar to bscorpion toxins [10]. Whole-cell recording showed that BmK AS could partially abolish Naþ currents in the NG108-15 cell line [16]. Moreover, BmK AS could * Corresponding author. Tel.: þ 86-21-54920300; fax: þ 86-2154920276. E-mail address: [email protected] (Y.-H. Ji).
promote noradrenaline release from the rat hippocampal slice [4]. All the results above suggest that BmK AS is an active polypeptide with multi-pharmacological functions. The B104 neuroblastoma cell line cloned from the rat central nervous system has been demonstrated to be excitable. Action potential-like responses in B104 cells are generated in a Naþ current-dependent manner [3]. The pharmacological properties and gating characteristic of voltage-gated Naþ currents in B104 cells are unique [2]. Though five alpha-subunits and beta 1-subunit of VGSCs mRNA have been detected by RT-PCR, it is still hard to understand the pharmacological and gating characteristics of VGSCs measured from undifferentiated B104 cells [1, 12]. In this study, we thus investigated the effects of BmK AS on the VGSCs in the B104 neuronal cell line. BmK AS were purified using previously described procedures [6]. The purity of the components was confirmed by means of either mass spectrometry or sequencing determination. Stocks of B104 cells were a gift from Dr D. Schubert (Salk Institute, San Diego, CA). The cells were cultured in Dulbecco’s modified Eagle’s medium (GIBCOBRL) containing 10% fetal bovine serum in glass flasks or 35 mm culture dishes (Corning Incorporated, Corning, NY). Flasks and dishes were maintained at 37 8C in an 5% CO2/95% air atmosphere and fed every fourth day.
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00094-6
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Recordings were performed using the whole cell mode of the patch clamp technique at room temperature. A holding potential of 2 100 mV was chosen. Membrane currents were measured with pipettes (4 – 6 MV) pulled from glass capillary tubes and connected to a patch clamp amplifier (EPC-9, HEKA Electronik, Lambrecht, Germany). The amplifier was operated with Pulse/PulseFit software (HEKA Electronik). The external solution was composed of (in mM): NaCl, 65; Choline –Cl, 50; TEACl, 20; KCl, 5; CaCl2, 0.01; MgCl2, 5; HEPES, 5; and D -glucose, 5. The pH was adjusted to 7.3– 7.4 with Tris– OH. The solution for the recording pipette was composed of (in mM): CsCl, 140; KCl, 5; CaCl2, 0.5; EGTA, 5; HEPES, 10; and Na-ATP, 3. The pH was adjusted to 7.3 –7.4 with CsOH. An Ag-AgCl wire was used with a saline bridge for the reference electrode. On-line leak subtraction was performed during recording using a P/4 protocol. The series resistance was partially compensated by 70– 80%. Tetrodotoxin (TTX) inhibition curves were fitted to a Langmuir binding isotherm of the form: f ðxÞ ¼ AX=ðkd þ XÞ where A represents a constant. The peak sodium conductance (gNa) at a different potential was calculated, as a chord conductance, from the corresponding peak current: gNa ¼ INa =ðV 2 VNa Þ where VNa is the sodium current reversal potential. Vg is the voltage for half-maximum activation, and kg is the slope factor. Vg and kg were determined from a least-squares fit to the data of a rising sigmoidal relationship: g=gmax ¼ 1=1 þ expððVg 2 VÞ=kg Þ The steady-state inactivation parameter (h1) 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 h1 ¼ 0:5. The slope factor of the h1 curve is termed kh at the mid-point (h1 ¼ 0:5). Vh and kh were determined from a least-squares fit to the data of the relationship: h1 ðI=Imax Þ ¼ 1=1 þ expððV 2 Vh Þ=kh Þ Statistical data are presented as the mean ^ standard errors. Significance is analyzed by t-test. Fig. 1A shows the average inhibition percentage of Naþ currents in each cell plotted against the TTX concentration. Only 9.3% of the Naþ currents were inhibited with 1 nM of TTX. Inhibition was not complete (80.5%) even at 10 mM TTX. The dose – response curve showed a plateau between 10 and 200 nM TTX. The dose –response curve was fitted by the sum of two Langmuir binding isotherm components, each progressing from zero inhibition to full inhibition over a three-decade domain as expected with values for halfmaximal TTX inhibition (IC50) of 1.95 ^ 0.92 and 794 ^ 1.44 nM, respectively. Therefore, 200 nM TTX
Fig. 1. Inhibition of tetrodotoxin in B104 cells. Naþ currents, elicited by depolarization from a holding potential of 2100 to 220 mV, were recorded before and after exposure to different concentration of tetrodotoxin (1, 10 and 100 nM and 1, 10 and 100 mM). (A) Currents from different experiments were normalized to those recorded in the absence of tetrodotoxin. An averaged inhibition of the current was plotted as a function of the tetrodotoxin concentration. Results are fitted by the sum of two Langmuir binding isotherms, each extending over a three-decade domain with kd of 89.1 ^ 8.37 and 794 ^ 1.44 nM, respectively. Numbers of experiments were from five to ten at different concentration ranges. (B) Currents recorded in a single B104 cell.
was suggested to distinguish the two components of Naþ current in B104 cells: TTX-sensitive (TTX-S) and TTXresistant (TTX-R) currents. The dose-dependent inhibitory effect of TTX on Naþ currents in a single B104 cell is shown in Fig. 1B. Similar to the results described by Gu et al. [2], the kinetics of Naþ currents showed a slight difference between currents inhibited by two group doses of TTX (1, 10 and 100 nM and 1, 10 and 100 mM), and the former was faster than the latter. Except for the subtle differences in kinetics, there was no significant variation between the voltage dependence of TTX-S and TTX-R currents (data not shown). The whole-cell recording in a single B104 cell found that the peak Naþ currents could be suppressed in the presence of 100 nM BmK AS, and partially recovered after washing (Fig. 2). As shown in Fig. 3, the whole Naþ currents and TTX-R components were insignificantly suppressed to 94 ^ 3.1 and 95 ^ 3.2% with 10 nM of BmK AS (P . 0:05). At dosages of 50, 100 and 500 nM, BmK AS dramatically (P , 0:01) suppressed the whole currents and TTX-R components to 83 ^ 2.6, 74 ^ 2.1, 78 ^ 3.9 and 86 ^ 2.7, 72 ^ 5.3, 79 ^ 2.7%, respectively. Furthermore, the differences between the dosages of 10 and 50 nM in both
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BmK AS at 500 nM could shift both steady-state activation and steady-state inactivation curves towards the hyperpolarizing direction without changing both slope factors (Fig. 4). For the steady-state activation curve, the mean value of Vg was shifted from 2 26.1 ^ 1.6 to 2 35.2 ^ 1.8 mV, while there seems to be no significant difference for the mean value of kg (from 9.2 ^ 1.1 to 8.9 ^ 1.0 mV) before and after application of BmK AS. For the steady-state inactivation curve, 500 nM BmK AS could move the mean value of V h from 2 76.0 ^ 0.4 to 2 90.9 ^ 0.5 mV. The change in kh (from 8.2 ^ 0.3 to 8.7 ^ 0.3 mV) was insignificant. It has been demonstrated that the pharmacological and gating characteristics of voltage-gated Naþ currents in B104 cells are unique [3]. There are two components, TTX-S and TTX-R, of the Naþ currents with different half-maximal inhibitions (IC50 ¼ 1:2 and 575.5 nM, respectively). The peak conductance –voltage relationship of whole currents has a V1/2 of 2 39.8 mV. V1/2 for steady-state inactivation is
Fig. 2. Inhibition of BmK AS on Naþ currents of B104 cells. A family of Naþ currents was recorded in a single B104 cell (A) before administration, (B) after administration and (C) after washing of 100 nM BmK AS. The holding potential was 2100 mV. Test pulses were applied for 50 ms in 10 mV increments from 280 to þ 70 mV.
whole currents and TTX-R currents were significant. At each dose, the difference between the inhibitory effects of BmK AS on the whole currents and TTX-R currents was not significant.
Fig. 3. Dose–response relationship of BmK AS on the whole Naþ currents (WC) and TTX-R component. Naþ currents were recorded by the same protocol as in Fig. 2. Peak Naþ currents were selected and normalized before and after exposure to different concentrations of BmK AS (1, 50, 100 and 500 nM). For TTX-R recording, 200 nM TTX was applied to block the TTX-S component. Data are the mean ^ SEM (N ¼ 6 – 10). * denotes a significant difference from the control (P , 0:01). # denotes a significant difference between 10 and 50 nM BmK AS (P , 0:05). The inset shows the inhibitory effects of BmK AS on TTX-R Naþ currents.
Fig. 4. Modulation of BmK AS on the steady-state activation and inactivation curves. The normalized conductance for g/gmax and h1 was determined as described in the text. (A) Averaged and normalized activation of Naþ currents in the absence (O) and in the presence of 500 nM BmK AS (K). The half-activation voltage (Vg) shifted from 226.1 ^ 1.6 to 235.2 ^ 1.8 mV, while the slope factor (kg) showed no significant change (from 9.2 ^ 1.1 to 8.9 ^ 1.0 mV). (B) Averaged and normalized steady-state inactivation of Naþ currents in the absence (†) and in the presence of 500 nM BmK AS (W). Measurements of steady-state inactivation were made following a standard two-pulse protocol. Cells were held at 2100 mV. A 500 ms prepulse ranging from 2150 to 250 mV (10 mV increments) was followed by a 50 ms test pulse at 220 mV. By exposure to 500 nM BmK AS, Vh was moved from 276.0 ^ 0.4 to 290.9 ^ 0.5 mV, and the mean value kh also showed no significant change (from 8.2 to 8.7 mV).
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2 81.6 mV. There is no difference in voltage dependence between TTX-S and TTX-R components. Similar results were found in our study, namely, there were two components of Naþ currents with distinct sensitivity to TTX, and the steady-state voltage dependence of the two currents was indistinguishable. A different point was that the proportion of the TTX-S component was smaller in this study. The results, the inhibition of BmK AS on both TTX-S and TTX-R components with similar potencies, indicated that the sensitivities of two components to BmK AS were similar. Both the steady-state activation and inactivation curves were shifted by BmK AS towards the negative direction which led to a change of the amplitude of Naþ currents. The shift of steady-state activation may increase the currents, whereas that of steady-state inactivation may decrease them. In the present study, it was found that Naþ currents were suppressed by BmK AS at several dosages, indicating that the shift of steady-state inactivation might contribute more to the amplitude of Naþ currents than the shift of steady-state activation. The inhibition of 500 nM BmK AS seemed to be less potent than that of 100 nM BmK AS on Naþ currents. It might reflect an integrated regulating effect of BmK AS on the amplitude of Naþ currents through modulating both steady-state activation and inactivation of VGSC. The proportion of 500 nM BmK AS affecting the amplitude of Naþ currents through modulating steady-state activation might be larger than that of 100 nM BmK AS. Taken together, the results suggest that BmK AS and other Naþ channel modulators derived from scorpion venom may be provided as useful tools for studying the Naþ currents in B104 cells, especially the TTX-R component.
Acknowledgements The authors thank Dr D. Schubert for B104 cells. This study was supported by the National Program of Basic Research of China (1999054001), partially by the Chinese Academy of Sciences, by grants from the National Nature Sciences Foundation of China (39625010), and by the Shanghai Metropolitan Fund for Research and Development (00JC14040).
References [1] S.D. Dib-Hajj, A.W. Hinson, J.A. Black, S.G. Waxman, Sodium channel mRNA in the B104 neuroblastoma cell line, FEBS Lett. 384 (1996) 78–82.
[2] X.Q. Gu, S. Dib-Hajj, M.A. Rizzo, S.G. Waxman, TTX-sensitive and -resistant Naþ currents, and mRNA for the TTX-resistant rH1 channel, are expressed in B104 neuroblastoma cells, J. Neurophysiol. 77 (1997) 236–246. [3] X.Q. Gu, S.G. Waxman, Action potential-like responses in B104 cells with low Naþ channel densities, Brain Res. 735 (1996) 50–58. [4] Y.H. Ji, H.Y. Huang, J.W. Zhang, M. Hoshino, T. Mochizuki, N. Yanaihara, BmK AS, an active scorpion polypeptide, enhances [3H]noradrenaline release from rat hippocampal slices, Biomed. Res. 18 (1997) 257 –260. [5] Y.H. Ji, Y.J. Li, J.W. Zhang, B.L. Song, T. Yamaki, T. Mochizuki, M. Hoshino, N. Yanaihara, Covalent structures of BmK AS and BmK AS-1, two novel bioactive polypeptides purified from Chinese scorpion Buthus martensi Karsch, Toxicon 37 (1999) 519 –536. [6] Y.H. Ji, P. Mansuelle, S. Terakawa, C. Kopeyan, N. Yanaihara, K. Xu, H. Rochat, Two neurotoxins (BmK I and BmK II) from the venom of scorpion Buthus martensi Karsch: purification, amino acid sequences and assessment of specific activity, Toxicon 34 (1996) 987 –1001. [7] Y.H. Ji, W.X. Wang, Q. Wang, Y.P. Huang, Biosensor binding of BmK ABT, a unique neurotoxic polypeptide on mammal brain and insect sodium channels, Eur. J. Pharmacol. 1 (2002) 25– 30. [8] A. Kuniyasu, S. Kawano, Y. Hirayama, Y.H. Ji, K. Xu, M. Ohkura, K. Furukawa, Y. Ohizumi, M. Hiraoka, H. Nakayama, A new scorpion toxin (BmK-PL) stimulates Ca2þ-release channel activity of the skeletal-muscle ryanodine receptor by an indirect mechanism, Biochem. J. 339 (1999) 343 –350. [9] Y.J. Li, Y.H. Ji, Binding characteristics of BmK I, an alpha-like scorpion neurotoxic polypeptide, on cockroach nerve cord synaptosomes, J. Pept. Res. 56 (2000) 195–200. [10] Y.J. Li, Y. Liu, Y.H. Ji, BmK AS: new scorpion neurotoxin binds to distinct receptor sites of mammal and insect voltage-gated sodium channels, J. Neurosci. Res. 61 (2000) 541–548. [11] M.F. Martin-Eauclaire, F. Couraud, Scorpion neurotoxins: effects of mechanisms, in: L.W. Chang, E.S. Dyer (Eds.), Handbook of Toxicology, Marcel Dekker, New York, 1995, pp. 683 –716. [12] Y. Oh, S.G. Waxman, Novel splice variants of the voltage-sensitive sodium channel alpha subunit, NeuroReport 9 (1998) 1267–1272. [13] L.D. Possani, B. Becerril, M. Delepierre, J. Tytgat, Scorpion toxins specific for Naþ-channels, Eur. J. Biochem. 264 (1999) 287 –300. [14] Z.Y. Tan, X. Mao, H. Xiao, Z.Q. Zhao, Y.H. Ji, Buthus martensi Karsch agonist of skeletal-muscle RyR-1, a scorpion active polypeptide: antinociceptive effect on rat peripheral nervous system and spinal cord, and inhibition of voltage-gated Naþ currents in dorsal root ganglion neurons, Neurosci. Lett. 2 (2001) 65–68. [15] Z.Y. Tan, H. Xiao, X. Mao, C.Y. Wang, Z.Q. Zhao, Y.H. Ji, The inhibitory effects of BmK IT2, a scorpion neurotoxin on rat nociceptive flexion reflex and a possible mechanism for modulating voltage-gated Naþ channels, Neuropharmacology 40 (2001) 352 –357. [16] Y. Wu, Y.H. Ji, Y.L. Shi, Sodium current in NG108-15 cell inhibited by scorpion BmK AS-1 and restored by its specific monoclonal antibodies, J. Nat. Toxins 10 (2001) 193 –198. [17] J.G. Ye, Z.Y. Tan, Y.J. Li, Y.P. Yan, C. Li, J. Chen, Y.H. Ji, Purification, cDNA cloning and functional assessment of BmK abT, a novel type neurotoxin from the scorpion species in the old world, FEBS Lett. 3 (2000) 136–140.
Modulation of BmK AS, a scorpion neurotoxic polypeptide, on voltagegated Naþ channels in B104 neuronal cell line Zhi-Yong Tan, Jin Chen, Hai-Ying Shun, Xing-Hua Feng, Yong-Hua Ji* The Key Laboratory of Neuroscience, Institute of Physiology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China Received 11 December 2002; received in revised form 6 January 2003; accepted 10 January 2003
Abstract The modulation of BmK AS, a neurotoxic polypeptide from scorpion Buthus martensi Karsch, on the voltage-gated Naþ channels has been investigated in the B104 neuroblastoma cell line by whole-cell patch clamping. It was found that the whole Naþ currents and tetrodotoxinresistant Naþ current were depressed in a biphasic manner and both steady-state activation and inactivation curves were shifted toward the hyperpolarizing direction in the presence of BmK AS. The results suggested that BmK AS could inhibit both tetrodotoxin-sensitive and resistant Naþ currents via modulating the gating mechanism of the voltage-gated Naþ channels in B104 cells. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Scorpion neurotoxin; BmK AS; B104; Voltage-gated Naþ channel
Most long-chain scorpion neurotoxic polypeptides, consisting of 60 –70 amino acid residues, are modulators of the voltage-gated Naþ channel (VGSC) in mammals, insects and other vertebrates [11]. According to biological specificity in vivo and pharmacological and electrophysiological activity, these scorpion neurotoxins can be divided into mammal-selective a or b toxins and excitatory or depressant insect-selective toxins [13]. The Asian scorpion Buthus martensi Karsch (BmK) is a species widely distributed in northwestern China, Mongolia and Korea. Up to now, at least ten long-chain neurotoxic components have been purified from BmK venom and determined to be modulators of Naþ channels [5 –7,9,14,15,17]. BmK AS is a unique long-chain polypeptide purified from the venom of B. martensi Karsch. It was found that BmK AS could enhance [3H]ryanodine binding to skeletaltype ryanodine receptors through an indirect mechanism [8], and bind to a distinct receptor site of VGSC on mammal and insect excitable cell membranes in a manner similar to bscorpion toxins [10]. Whole-cell recording showed that BmK AS could partially abolish Naþ currents in the NG108-15 cell line [16]. Moreover, BmK AS could * Corresponding author. Tel.: þ 86-21-54920300; fax: þ 86-2154920276. E-mail address: [email protected] (Y.-H. Ji).
promote noradrenaline release from the rat hippocampal slice [4]. All the results above suggest that BmK AS is an active polypeptide with multi-pharmacological functions. The B104 neuroblastoma cell line cloned from the rat central nervous system has been demonstrated to be excitable. Action potential-like responses in B104 cells are generated in a Naþ current-dependent manner [3]. The pharmacological properties and gating characteristic of voltage-gated Naþ currents in B104 cells are unique [2]. Though five alpha-subunits and beta 1-subunit of VGSCs mRNA have been detected by RT-PCR, it is still hard to understand the pharmacological and gating characteristics of VGSCs measured from undifferentiated B104 cells [1, 12]. In this study, we thus investigated the effects of BmK AS on the VGSCs in the B104 neuronal cell line. BmK AS were purified using previously described procedures [6]. The purity of the components was confirmed by means of either mass spectrometry or sequencing determination. Stocks of B104 cells were a gift from Dr D. Schubert (Salk Institute, San Diego, CA). The cells were cultured in Dulbecco’s modified Eagle’s medium (GIBCOBRL) containing 10% fetal bovine serum in glass flasks or 35 mm culture dishes (Corning Incorporated, Corning, NY). Flasks and dishes were maintained at 37 8C in an 5% CO2/95% air atmosphere and fed every fourth day.
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00094-6
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Recordings were performed using the whole cell mode of the patch clamp technique at room temperature. A holding potential of 2 100 mV was chosen. Membrane currents were measured with pipettes (4 – 6 MV) pulled from glass capillary tubes and connected to a patch clamp amplifier (EPC-9, HEKA Electronik, Lambrecht, Germany). The amplifier was operated with Pulse/PulseFit software (HEKA Electronik). The external solution was composed of (in mM): NaCl, 65; Choline –Cl, 50; TEACl, 20; KCl, 5; CaCl2, 0.01; MgCl2, 5; HEPES, 5; and D -glucose, 5. The pH was adjusted to 7.3– 7.4 with Tris– OH. The solution for the recording pipette was composed of (in mM): CsCl, 140; KCl, 5; CaCl2, 0.5; EGTA, 5; HEPES, 10; and Na-ATP, 3. The pH was adjusted to 7.3 –7.4 with CsOH. An Ag-AgCl wire was used with a saline bridge for the reference electrode. On-line leak subtraction was performed during recording using a P/4 protocol. The series resistance was partially compensated by 70– 80%. Tetrodotoxin (TTX) inhibition curves were fitted to a Langmuir binding isotherm of the form: f ðxÞ ¼ AX=ðkd þ XÞ where A represents a constant. The peak sodium conductance (gNa) at a different potential was calculated, as a chord conductance, from the corresponding peak current: gNa ¼ INa =ðV 2 VNa Þ where VNa is the sodium current reversal potential. Vg is the voltage for half-maximum activation, and kg is the slope factor. Vg and kg were determined from a least-squares fit to the data of a rising sigmoidal relationship: g=gmax ¼ 1=1 þ expððVg 2 VÞ=kg Þ The steady-state inactivation parameter (h1) 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 h1 ¼ 0:5. The slope factor of the h1 curve is termed kh at the mid-point (h1 ¼ 0:5). Vh and kh were determined from a least-squares fit to the data of the relationship: h1 ðI=Imax Þ ¼ 1=1 þ expððV 2 Vh Þ=kh Þ Statistical data are presented as the mean ^ standard errors. Significance is analyzed by t-test. Fig. 1A shows the average inhibition percentage of Naþ currents in each cell plotted against the TTX concentration. Only 9.3% of the Naþ currents were inhibited with 1 nM of TTX. Inhibition was not complete (80.5%) even at 10 mM TTX. The dose – response curve showed a plateau between 10 and 200 nM TTX. The dose –response curve was fitted by the sum of two Langmuir binding isotherm components, each progressing from zero inhibition to full inhibition over a three-decade domain as expected with values for halfmaximal TTX inhibition (IC50) of 1.95 ^ 0.92 and 794 ^ 1.44 nM, respectively. Therefore, 200 nM TTX
Fig. 1. Inhibition of tetrodotoxin in B104 cells. Naþ currents, elicited by depolarization from a holding potential of 2100 to 220 mV, were recorded before and after exposure to different concentration of tetrodotoxin (1, 10 and 100 nM and 1, 10 and 100 mM). (A) Currents from different experiments were normalized to those recorded in the absence of tetrodotoxin. An averaged inhibition of the current was plotted as a function of the tetrodotoxin concentration. Results are fitted by the sum of two Langmuir binding isotherms, each extending over a three-decade domain with kd of 89.1 ^ 8.37 and 794 ^ 1.44 nM, respectively. Numbers of experiments were from five to ten at different concentration ranges. (B) Currents recorded in a single B104 cell.
was suggested to distinguish the two components of Naþ current in B104 cells: TTX-sensitive (TTX-S) and TTXresistant (TTX-R) currents. The dose-dependent inhibitory effect of TTX on Naþ currents in a single B104 cell is shown in Fig. 1B. Similar to the results described by Gu et al. [2], the kinetics of Naþ currents showed a slight difference between currents inhibited by two group doses of TTX (1, 10 and 100 nM and 1, 10 and 100 mM), and the former was faster than the latter. Except for the subtle differences in kinetics, there was no significant variation between the voltage dependence of TTX-S and TTX-R currents (data not shown). The whole-cell recording in a single B104 cell found that the peak Naþ currents could be suppressed in the presence of 100 nM BmK AS, and partially recovered after washing (Fig. 2). As shown in Fig. 3, the whole Naþ currents and TTX-R components were insignificantly suppressed to 94 ^ 3.1 and 95 ^ 3.2% with 10 nM of BmK AS (P . 0:05). At dosages of 50, 100 and 500 nM, BmK AS dramatically (P , 0:01) suppressed the whole currents and TTX-R components to 83 ^ 2.6, 74 ^ 2.1, 78 ^ 3.9 and 86 ^ 2.7, 72 ^ 5.3, 79 ^ 2.7%, respectively. Furthermore, the differences between the dosages of 10 and 50 nM in both
Z.-Y. Tan et al. / Neuroscience Letters 340 (2003) 123–126
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BmK AS at 500 nM could shift both steady-state activation and steady-state inactivation curves towards the hyperpolarizing direction without changing both slope factors (Fig. 4). For the steady-state activation curve, the mean value of Vg was shifted from 2 26.1 ^ 1.6 to 2 35.2 ^ 1.8 mV, while there seems to be no significant difference for the mean value of kg (from 9.2 ^ 1.1 to 8.9 ^ 1.0 mV) before and after application of BmK AS. For the steady-state inactivation curve, 500 nM BmK AS could move the mean value of V h from 2 76.0 ^ 0.4 to 2 90.9 ^ 0.5 mV. The change in kh (from 8.2 ^ 0.3 to 8.7 ^ 0.3 mV) was insignificant. It has been demonstrated that the pharmacological and gating characteristics of voltage-gated Naþ currents in B104 cells are unique [3]. There are two components, TTX-S and TTX-R, of the Naþ currents with different half-maximal inhibitions (IC50 ¼ 1:2 and 575.5 nM, respectively). The peak conductance –voltage relationship of whole currents has a V1/2 of 2 39.8 mV. V1/2 for steady-state inactivation is
Fig. 2. Inhibition of BmK AS on Naþ currents of B104 cells. A family of Naþ currents was recorded in a single B104 cell (A) before administration, (B) after administration and (C) after washing of 100 nM BmK AS. The holding potential was 2100 mV. Test pulses were applied for 50 ms in 10 mV increments from 280 to þ 70 mV.
whole currents and TTX-R currents were significant. At each dose, the difference between the inhibitory effects of BmK AS on the whole currents and TTX-R currents was not significant.
Fig. 3. Dose–response relationship of BmK AS on the whole Naþ currents (WC) and TTX-R component. Naþ currents were recorded by the same protocol as in Fig. 2. Peak Naþ currents were selected and normalized before and after exposure to different concentrations of BmK AS (1, 50, 100 and 500 nM). For TTX-R recording, 200 nM TTX was applied to block the TTX-S component. Data are the mean ^ SEM (N ¼ 6 – 10). * denotes a significant difference from the control (P , 0:01). # denotes a significant difference between 10 and 50 nM BmK AS (P , 0:05). The inset shows the inhibitory effects of BmK AS on TTX-R Naþ currents.
Fig. 4. Modulation of BmK AS on the steady-state activation and inactivation curves. The normalized conductance for g/gmax and h1 was determined as described in the text. (A) Averaged and normalized activation of Naþ currents in the absence (O) and in the presence of 500 nM BmK AS (K). The half-activation voltage (Vg) shifted from 226.1 ^ 1.6 to 235.2 ^ 1.8 mV, while the slope factor (kg) showed no significant change (from 9.2 ^ 1.1 to 8.9 ^ 1.0 mV). (B) Averaged and normalized steady-state inactivation of Naþ currents in the absence (†) and in the presence of 500 nM BmK AS (W). Measurements of steady-state inactivation were made following a standard two-pulse protocol. Cells were held at 2100 mV. A 500 ms prepulse ranging from 2150 to 250 mV (10 mV increments) was followed by a 50 ms test pulse at 220 mV. By exposure to 500 nM BmK AS, Vh was moved from 276.0 ^ 0.4 to 290.9 ^ 0.5 mV, and the mean value kh also showed no significant change (from 8.2 to 8.7 mV).
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2 81.6 mV. There is no difference in voltage dependence between TTX-S and TTX-R components. Similar results were found in our study, namely, there were two components of Naþ currents with distinct sensitivity to TTX, and the steady-state voltage dependence of the two currents was indistinguishable. A different point was that the proportion of the TTX-S component was smaller in this study. The results, the inhibition of BmK AS on both TTX-S and TTX-R components with similar potencies, indicated that the sensitivities of two components to BmK AS were similar. Both the steady-state activation and inactivation curves were shifted by BmK AS towards the negative direction which led to a change of the amplitude of Naþ currents. The shift of steady-state activation may increase the currents, whereas that of steady-state inactivation may decrease them. In the present study, it was found that Naþ currents were suppressed by BmK AS at several dosages, indicating that the shift of steady-state inactivation might contribute more to the amplitude of Naþ currents than the shift of steady-state activation. The inhibition of 500 nM BmK AS seemed to be less potent than that of 100 nM BmK AS on Naþ currents. It might reflect an integrated regulating effect of BmK AS on the amplitude of Naþ currents through modulating both steady-state activation and inactivation of VGSC. The proportion of 500 nM BmK AS affecting the amplitude of Naþ currents through modulating steady-state activation might be larger than that of 100 nM BmK AS. Taken together, the results suggest that BmK AS and other Naþ channel modulators derived from scorpion venom may be provided as useful tools for studying the Naþ currents in B104 cells, especially the TTX-R component.
Acknowledgements The authors thank Dr D. Schubert for B104 cells. This study was supported by the National Program of Basic Research of China (1999054001), partially by the Chinese Academy of Sciences, by grants from the National Nature Sciences Foundation of China (39625010), and by the Shanghai Metropolitan Fund for Research and Development (00JC14040).
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