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Electrophysiological characterization of BmK I, an α-like scorpion toxin, on rNav1.5 expressed in HEK293t cells
Toxicology in Vitro 22 (2008) 1582–1587
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Electrophysiological characterization of BmK I, an a-like scorpion toxin, on rNav1.5 expressed in HEK293t cells Xing-Hua Feng a, Jue-Xu Chen b, Ying Liu a, Yong-Hua Ji b,* a b
Graduate School of the Chinese Academy of Sciences, Shanghai Institute of Physiology, Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China School of Life Sciences of Shanghai University, Shang-Da Road 99, Shanghai 200444, PR China
a r t i c l e
i n f o
Article history: Received 30 March 2008 Accepted 23 June 2008 Available online 28 June 2008 Keywords: HEK293t cell Cardiac sodium channels BmK I Patch clamp
a b s t r a c t A recent study described the pharmacological properties of BmK I, an a-like toxin from the Chinese scorpion Buthus martensi Karsch, on the cardiac sodium channel (hH1) expressed in Xenopus oocytes. Considering that a-like toxins are unique in their inability to bind to rat synaptosomes despite a high toxicity by intravenous injection, the present study investigated the pharmacological properties of BmK I on rNav1.5 expressed in a mammalian HEK293t cell line. The results include: (1) BmK I slowed and partially inhibited the inactivation of rNav1.5, produced a substantial persistent current and increased peak current (the EC50 for increasing peak current by BmK I was 99.4 ± 20.1 nM); (2) BmK I delayed the recovery of the sodium channel from inactivation; (3) after exposure to 300 nM BmK I, the steady-state activation curve of rNav1.5 was negatively shifted by about 19 mV; and (4) the association of BmK I and rNav1.5 was faster than their dissociation. The results show that BmK I displayed the pharmacological characteristics of an a-like toxin on rNav1.5 channels expressed in HEK293t cells, and suggested that the host expression system should be taken into consideration when characterizing the pharmacological properties of toxins. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Voltage-gated sodium channels (VGSCs) respond to the depolarizing drive associated with the generation of action potentials in most excitable cells. In vivo, VGSCs usually consist of a large pore-forming a-subunit and one or more auxiliary subunits. The a-subunit has a highly conserved structure composed of four homologous domains (DI-DIV), each with six transmembrane segments (S1–S6) and a hairpin-like pore region between S5 and S6. Due to the structural conservation and their functional importance in cellular excitability (Yu and Catterall, 2003; Zlotkin, 1999), VGSCs are targeted by a variety of toxins, including peptides from scorpion venom (Wang and Wang, 2003). Most scorpion neurotoxins targeting VGSCs are single chain polypeptides composed of 60–70 amino acids cross-linked by four disulfide bridges (Arnon et al., 2005; Chen et al., 2002; Possani et al., 1999). These neurotoxic polypeptides comprise two main groups: a- and b-toxins (Gordon et al., 1996; Leipold et al., 2004). Scorpion
Abbreviations: VGSCs, voltage-gated sodium channels; BmK, Buthus martensi Karsch; EC50, half maximal effective concentration; HEK, human embryonic kidney; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; BSA, bovine serum albumin; HEPES, (N-(2-hydroxyethyl) piperazine-N0 -(2-ethanesulfonic acid)). * Corresponding author. Tel./fax: +86 21 66135189. E-mail addresses: [email protected], [email protected], [email protected] (Y.-H. Ji). 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.06.009
a-toxins bind to site 3, which is occupied by several groups of polypeptide: a-scorpion toxins, sea-anemone toxins and some spider toxins. Scorpion a-toxins prolong the action potential by slowing the inactivation of VGSCs. According to the different pharmacological and binding properties, the a-toxins can be further divided into three subgroups: classical a-, a-like, and insect a-toxins. Classical a-toxins are highly toxic to mammals, whereas the insect a-toxins are highly toxic to insects. The a-like toxins act on both mammals and insects, but they are unique in their inability to bind to rat synaptosomes despite a high toxicity by intravenous injection (Cestele et al., 1999; Gilles et al., 1999; Sautiere et al., 1998). BmK I is an a-like toxin from the eastern Asian scorpion Buthus martensi Karsch (Ji et al., 1996). BmK I acts on VGSCs in the central and peripheral nervous systems, thus producing epileptic seizures and hyperalgesia (Bai et al., 2006; Chen et al., 2001). Interestingly, BmK I also functions as a cardiotoxin: it increases rat heart contractility and prolongs inactivation of sodium currents in isolated rat ventricular myocytes (Sun et al., 2003; Sun et al., 2005). Since Nav1.5 is the predominant isoform expressed in heart muscle (Gellens et al., 1992), BmK I was postulated to be active on this cardiac sodium channel isoform. Consistent with this speculation, the inactivation of hNav1.5 expressed in Xenopus oocytes could be slowed down by BmK I (Goudet et al., 2001). It has been reported, however, that VGSCs expressed in Xenopus oocytes exhibit different electrophysiological properties (e.g., the slow mode of gating) compared to in vivo or when mammalian cells are used as expression systems. These differences might
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further influence pharmacological effects of toxins targeting VGSCs. Therefore, in the present paper, we studied the pharmacological effects of the presumptive cardiotoxin on a subunit of cardiac sodium channel isoform-rNav1.5, expressed in a mammalian HEK293t cell line. 2. Material and methods 2.1. Material BmK venom collected by electric stimulation was purchased from a scorpion farm in Zhengzhou, Henan province, China. BmK I was purified according to the method described by Ji et al. (1996). The purity of this component was confirmed by means of either mass spectrometry or sequencing determination. HEK293T cell line was from Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Unless otherwise stated, all chemicals were from Sigma. 2.2. Cell culture and transient transfection HEK293t cells were maintained in DMEM (GIBCO BRL) supplemented with 10% FBS, 20 lg/ml penicillin, and 10 lg/ml streptomycin in a 5% CO2 incubator. The cell line was transfected with plasmids coding a subunit of rat cardiac sodium channel (rNav1.5) by means of Lipofectamine2000 (Invitrogen) reagent according to the instructions of the manufacturer. Briefly, HEK293t cells were passaged onto 35 mm culture plates at about 25% confluence. Following incubation for 24 h, cells were exposed to a 2 ml Opti-MEM medium (GIBCO Life Technologies) containing 2 ll Lipofectamine2000 and 1–2 lg sodium channel expression vector. Following a 5–24 h incubation at 37 °C with humidified 5% CO2, the medium was exchanged for normal HEK293t cell growing medium. A post-transfection incubation for 36–48 h preceded the electrophysiological recordings. Cells with inward currents were deemed as transfection-positive ones. Whole-cell patch clamp recordings were conducted at room temperature using an Axopatch-200B amplifier (Axon Instruments Inc., Union, CA). Electrophysiological recordings were started 10 min after establishing a whole-cell configuration. To eliminate the effect of steady-state inactivation during experiments, the holding potential was set to 140 mV. The series resistance was compensated (70–80%) to minimize voltage errors. Leak subtraction was performed using a P/4 protocol applied before the test protocol for all voltage clamp recordings. Signals were low-pass filtered at 5 kHz and digitized at 20 kHz. We used patch pipettes that had a resistance of 2–3 MX when filled with the pipette solution listed below. The pipette solution contained (mM): CsF 140, CsCl 10, MgCl2 1, EGTA 2, HEPES 10 (pH 7.3). The bath solution contained (mM): NaCl 140, MgCl2 1, CaCl2 2, HEPES 10, Glucose 10 (pH 7.3). 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 BSA alone did not alter sodium 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 lm from the cells. After 10-min perfusion with the toxin, step pulses as detailed in the figure legends were used to investigate the effect of BmK I on rNav1.5 channels. 2.3. Data analysis Conductance was calculated as G(V) = Ipeak(V)/(V Erev), where the reversal potential (Erev) was measured experimentally for each cell.
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The voltage dependence of activation was quantified by fitting the conductance measures to a Boltzmann function: G(V) = Gmax/ [1 + exp (Vtest V1/2)/K]. Steady-state inactivations were fitted to a Boltzmann function with a non-zero pedestal (I0) calculated as I/Imax = (1 I0)/ [1 + exp (V1/2 Vtest)/K] + I0, where V1/2 was the test potential at which the channels were half-maximally activated and K was the slope factor. The data in Figs. 1B, 2 and 4C were fitted with a single exponential function: y = A exp (t/s) + y0, where s was the time constant. The equation used for fitting dose–response relationships was Inorm = A/[1 + ([BmK I]/EC50)p] + C, where Inorm was the measured and normalized peak current, EC50 was the half maximal effective concentration, and p was the slope factor. Data were analyzed using the Clampfit8.2 (Axon Instruments, USA) and Origin6.0 (OriginLab Corp., Northampton, MA, USA), and they were presented as means ± SEM. The number of cells examined was represented by n. Student’s paired or unpaired t-tests were used for comparisons, and statistical significance was assessed as P < 0.05.
3. Results 3.1. Effects of BmK I on rNav1.5 sodium currents Representative sodium currents recorded from a HEK293t cell expressing rNav1.5 in the absence and in the presence of 300 nM BmK I were shown in Fig. 1A, which shows that BmK I strongly slowed down the inactivation of sodium currents and caused persistent currents. Under control conditions, inactivation increased with increasing voltage, which was maximal at around 10 mV. After application of 300 nM BmK I, inactivation was strongly impaired. Comparison of time constants for inactivation in control and in the presence of BmK I shows that inactivation was very significantly slowed over potentials from 40 to 0 mV (Fig. 1B). The relative persistent currents were obtained by dividing corresponding peak currents (Ipeak) by persistent currents (Ipersistent). In the absence of BmK I, there were only very small relative persistent currents; in the presence of 300 nM BmK I, relative persistent currents increased with increasing depolarizations. Comparison with control shows that BmK I significantly induced persistent currents at potentials more positive than 30 mV (Fig. 1C). The corresponding I–V curves were shown in Fig. 1D. It indicates that BmK I significantly increased the peak current over the range from 50 to +10 mV. Moreover, this effect of BmK I on peak currents was dose-dependent (Fig. 1E). After 10-min treatment with different concentration of BmK I, currents were elicited by 70-ms depolarizations to 30 mV from the holding potential of 140 mV. The increase for relative increment peak current enhanced from 3% by 30 nM BmK I (n = 5) to 18% after exposure to 100 nM BmK I (n = 5). The relative increment was approximately 46% and 43% in the presence of 300 nM (n = 4) and 1000 nM (n = 5) BmK I, respectively. The EC50 value determined by a sigmoidal fit of the data in Fig. 1E was 99.4 ± 20.1 nM. 3.2. BmK I slowed the recovery of rNav1.5 channels Because BmK I impaired the inactivation of rNav1.5 channels, we further investigated its effect on the recovery from inactivation (Fig. 2). In the absence and presence of 300 nM BmK I, recovery increased with intervals at 140 mV. Under control conditions, the recovery of rNav1.5 was fast, with the time constant of 2.31 ± 0.32 ms. After treatment with 300 nM BmK I, the recovery
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Fig. 1. Functional effect of BmK I on rNav1.5 currents. (A) Current responses to test pulses for rNav1.5 channels under control conditions (left) and after application of 300 nM BmK I (right). 70-ms test pulses ranged from 100 mV to +40 mV by a 10-mV increment from the holding potential of 140 mV. For clarity, only the first 40 ms of the currents are shown. (B) Effect of 300 nM BmK I on the relative persistent currents, here expressed as Ipersistent (% Ipeak). Persistent currents (Ipersitent) were measured as the mean current remaining between 45 and 65 ms after the start of depolarizations. (C) Current–voltage (I–V) curves of sodium currents in the absence (closed squares) and presence of 300 nM BmK I (closed circles). (n = 6); *P < 0.05. (D) Time constants for inactivation (Tau) in the absence (closed squares) and presence of 300 nM BmK I (closed squares). The time constants were obtained by fitting the declining phase of sodium currents with single exponential functions. The holding potential was 140 mV. n = 5; ** P < 0.01. (E) Dose-dependent enhancement of the peak rNav1.5 current induced by BmK I. After 10-min treatment with different concentration of BmK I, currents were elicited by 70-ms depolarizations to 30 mV from the holding potential of 140 mV. Relative increments of peak currents were calculated by dividing the increments induced by BmK I with the peak current under control, and plotted against the concentration of BmK I. The EC50 value determined by a sigmoidal fit is 99.4 ± 20.1 nM.
was significantly slowed down, with the time constant of 5.36 ± 0.51 ms. In addition, 300 nM BmK I decreased the percentage of available sodium channels after 30-ms recovery at 140 mV. The fraction recovery of rNav1.5 dropped from 93% (control) to 84% (300 nM BmK I). This indicates that, in the presence of BmK I, more sodium channels were in an inactivation state, requiring more time to recover. 3.3. BmK I enhanced the voltage-dependent activation of rNav1.5 channels BmK I could induce rNav1.5 currents at more negative test voltage, which can be deduced from Fig. 1D. This caused a dose-depen-
dent, hyperpolarizing shift in the steady-state activation curve (Fig. 3A). Under control conditions, the half-activation voltage (V1/2) of rNav1.5 was 36.85 ± 1.43 mV. In the presence of 30 nM BmK I (n = 4), the shift of V1/2 was 5.06 ± 1.64 mV. The shift by 100 nM (n = 7) and 300 nM (n = 5) BmK I was 10.22 ± 1.22 mV and 18.78 ± 2.45 mV, respectively. There was, however, no significant difference between slopes of these curves. To study steady-state inactivation, cells were held for 400 ms at prepulse potentials ranging from 140 to +20 mV and then subjected to a depolarizing pulse of 30 mV for 40 ms. The steady-state inactivation curves in the absence and presence of BmK I were displayed in Fig. 3B. Statistical analyses show that there was no significant effect of BmK I on steady-state inactivation.
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Fig. 2. Effect of BmK I on the recovery kinetics of rNav1.5 channels. To measure recovery from inactivation, a 30-ms conditioning step to 30 mV was applied and then availability was assayed with a test step to 30 mV after variable recovery intervals at 140 mV. Each step pair was delivered at a frequency of 0.2 Hz. Fraction of available channels was calculated as the ratio of the test to the conditioning current amplitude and was plotted versus recovery interval in absence (closed squares) and presence of 300 nM BmK I (closed circles). Data were fitted with single exponentials. Control: n = 9; 300 nM BmK I: n = 6. *P < 0.05.
3.4. Kinetics of BmK I acting on rNav1.5 channels To investigate the kinetics of BmK I acting on rNav1.5 channels, the association and disassociation rat of BmK I and rNav1.5 channels was studied. Representative traces for association were shown in Fig. 4A, and those for dissociation in Fig. 4B. Comparison of the time constants from Fig. 4C shows that the binding process of BmK I, with time constant of 21.48 ± 1.68 s was much faster than that of unbinding, with time constant of 73.09 ± 17.19 s. Two traces shown in Fig. 4D were the first current in Fig. 4A (before application of 300 nM BmK I) and the last one in Fig. 4B (after a 10-min washout), respectively. There was nearly no difference between them, which suggests that the effect of BmK I on rNav1.5 was almost completely reversible within the time span of the experiment. 4. Discussion In this study, BmK I was demonstrated to slow down the inactivation of sodium currents and induce persistent currents, which indicates that BmK I is active on the rNav1.5 transiently expressed in HEK293t, reinforcing the previous conclusions that BmK I is an a-like toxin and that cardiac sodium channels are one of the targets of BmK I. The intracellular linker between DIII and DIV, in which the critical motif is the hydrophobic triad IFM, and the S4 in DIV, which is in juxtaposition with the receptor site of a-like scorpion toxins (DIV S3–S4) (Rogers et al., 1996), are both involved in the fast inactivation of sodium channels (Chen et al., 1996; Eaholtz et al., 1994; Eaholtz et al., 1998; Kontis and Goldin, 1997). Thus, it was believed that the binding of a-like scorpion toxins (including BmK I) hampered the movement of DIV S4 (Sheets and Hanck, 1995) required for the formation of binding site for IFM, resulting in the slowing the formation of receptor site for IFM, so the inactivation was slowed down. Moreover, in the presence of BmK I, the receptor site for IFM formed in response to depolarizations had less affinity, which leaded to the equilibrium between bound and unbound states more partially favoring the unbound state, which resulted in persistent currents. BmK I delayed the recovery of rNav1.5 channels from inactivation. Moreover, our study indicates that, in the presence of BmK I,
Fig. 3. Effects of BmK I on the gating of the rNav1.5 channel. (A) Effect of BmK I on the steady-state activation of rNav1.5. Steady-state activation was studied by measuring the peak sodium conductance during a 70-ms test pulse to various test potentials, in 10-mV increment, from a 140 mV holding voltage. Normalized conductance (expressed as the conductance relative to the maximum conductance G/Gmax) was plotted against test voltages and fitted with Boltzmann functions. The lines showed the fit of the means of control (solid line), 10 (dashed line), 100 (dotted line) and 300 nM (dash dot line) BmK I (n = 5), respectively. (B) Effect of BmK I on the steady-state inactivation of rNav1.5. Steady-state inactivation curves were determined using 400-ms conditioning pulses to voltages between 140 and +20 mV and a standard 40-ms test pulse to 30 mV. Normalized test currents were plotted against the conditioning voltages and fitted with Boltzmann functions. The lines showed the fit of the means of control (solid line), 10 (dashed line), 100 (dotted line) and 300 nM (dash dot line) BmK I, respectively. Control: n = 5; 10 nM BmK I: n = 4; 100 nM BmK I: n = 7; 300 nM BmK I: n = 5.
more sodium channels were in another inactivation state (84% vs. 93% channel available after 30-ms recovery at 140 mV), which needed more time to recover. As such, it was assumed that, under control conditions, the recovery from fast inactivation was dominant. In the presence of BmK I, after stimulation, more sodium channels were in the inactivation state requiring more recovery time, and this made a significant contribution to total recovery. Thus, the recovery was slowed down. This effect of BmK I was in accordance with result obtained by Chen and coworkers, who demonstrated that an a-like toxin, Lqh III, slowed down the recovery rate from inactivation of cardiac sodium channel hH1 (Chen and Heinemann, 2001). BmK I induced a shift of the steady-state activation curve to more negative potentials, whereas voltage dependence of steady-
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Fig. 4. Association and disassociation kinetics of BmK I on rNav1.5 channels. (A) Representative recording of association obtained during exposure to 300 nM BmK I. (B) Representative recordings of dissociation during washout with bath solution. Representative currents were elicited with ten 90-ms pulses applied from 140 mV to 30 mV every 30 seconds. The first trace of association (dissociation) was recorded immediately once applying 300 nM BmK I (bath solution). The holding potential was 140 mV. The vertical lines indicate 10 ms after the test pulses start. (C) The association and disassociation rate of BmK I and rNav1.5. The association and dissociation rate were estimated by the normalized sodium currents at 10 ms after the test pulse start (I10 ms/Imax at 10 ms). Normalized sodium currents of association (closed squares) and dissociation (closed circles) were plotted against the application time and fitted by single exponential functions. The time constants of association and dissociation were estimated to be 21.48 ± 1.68 s and 73.09 ± 17.19 s, respectively. (D) Two superimposed currents. The current with mark ‘‘control” was the first trace in Fig. 4A and that with ‘‘wash” was the last one in Fig. 4B (n = 5).
state inactivation was not affected. This finding was in agreement with that obtained on TTX-sensitive sodium currents recording from DRGs (Chen et al., 2005) and with findings from research of LqhIII on skeletal muscle sodium channels expressed in HEK293t cells (Chen et al., 2000). These results might imply a common mode of action for a-like scorpion toxins on the gating properties of sodium channels in vivo resulting probably from a common binding site for these polypeptide neurotoxins, which warranted further investigation. It is noteworthy that, in contrast to our results, Goudet et al. reported that BmK I induced a hyperpolarizing shift of the steady-state inactivation of the human cardiac sodium channels expressed in Xenopus oocytes (Goudet et al., 2001). This discrepancy was postulated to result from the different host cells. It was highly suspected that the auxiliary b1-subunit, stably expressed by HEK293t cells (Moran et al., 2000) but not in oocytes, and the different lipid content (Martens et al., 2000, 2001) might be involved in the discrepancy. Other possibilities cannot, however, be ruled out. Our work shows that association of BmK I to cardiac sodium channels was more rapid than dissociation. Moreover, in our study, it was observed that the effect of BmK I was only partially reversible (data not shown), which was also reported in DRGs (Chen et al., 2005). The result in Fig. 4D, however, seems to show that the effect of BmK I could be almost completely reversible. Since site 3 toxins bind with sodium channels in a voltage-dependent manner and depolarization induced a dramatic decrease of a-scorpion toxins affinity to sodium channels (Chen et al., 2000; Jover et al., 1980; Mozhayeva et al., 1980), this discrepancy should be the result of the repetitive long test pulses during washout, which is also indirect evidence for voltage-dependent binding of BmK I to rNav1.5.
Nav1.5 was the predominant sodium channel subtype in heart muscles (Gellens et al., 1992), and played a key role in the cardiac excitability and conduction. Since the important role of normal Nav1.5 function in cardiac rhythm had been highlighted by studies characterizing the mutant resulting in LQT3 and Brugada syndrome (Dumaine et al., 1996; Veldkamp et al., 2000; Wang et al., 1995), it was suggested that BmK I-induced inactivation-slowing and persistent current were at least partly responsible for cardiac arrhythmia induced by exposure of isolated rat hearts to BmK I (Sun et al., 2003). Moreover, an increase in the amplitude and voltage range of the so-called sodium ‘‘window current”; i.e., the maintained inward sodium current caused by an overlap in activation and inactivation relations, resulting from the negative shift of voltage-dependent activation, might also partially account for cardiac arrhythmia induced by BmK I. Furthermore, as a consequence of inactivation impairment, persistent current and a larger window current in the presence of BmK I, sodium channels could conduct much more inward sodium current than in the absence of the toxin. The sodium gradient becomes insufficient to maintain the calcium gradient, so intracellular calcium increases through sodium–calcium exchange (Han et al., 2002; Litwin et al., 1998; Noble and Noble, 2006). This might explain the increase in rat heart contractility induced by BmK I. Finally, the slower recovery from inactivation decreases the density of available cardiac sodium channels during the cardiac cycle, which is thought to be one cause of arrhythmogenesis in patients with Brugada and Long-QT syndrome (Clancy and Rudy, 2002). In conclusion, the present study shows that the scorpion toxin BmK I exhibited a-like toxin properties on rNav1.5 expressed in HEK293t cells. BmK I impaired inactivation processes and induced substantial persistent sodium currents. Moreover, BmK I slowed
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down the recovery from inactivation and increased peak currents (the EC50 for increasing peak current by BmK I was 99.4 ± 20.1 nM). In addition, BmK I modified the gating properties by shifting the steady-state activation in the hyperpolarization direction. Acknowledgements Authors thank Kaoru Yamaoka for his generous gift of rNav1.5 and Zhi-Yong Tan for proofreading. This study was supported by National Program of Basic Research of China (2006CB500801). References Arnon, T., Potikha, T., Sher, D., Elazar, M., Mao, W., Tal, T., Bosmans, F., Tytgat, J., BenArie, N., Zlotkin, E., 2005. BjalphaIT: a novel scorpion alpha-toxin selective for insects – unique pharmacological tool. Insect Biochemistry and Molecular Biology 35 (3), 187–195. Bai, Z.T., Zhao, R., Zhang, X.Y., Chen, J., Liu, T., Ji, Y.H., 2006. The epileptic seizures induced by BmK I, a modulator of sodium channels. Experimental Neurology 197 (1), 167–176. Cestele, S., Stankiewicz, M., Mansuelle, P., De Waard, M., Dargent, B., Gilles, N., Pelhate, M., Rochat, H., Martin-Eauclaire, M.F., Gordon, D., 1999. Scorpion alphalike toxins, toxic to both mammals and insects, differentially interact with receptor site 3 on voltage-gated sodium channels in mammals and insects. European Journal of Neuroscience 11 (3), 975–985. Chen, H., Heinemann, S.H., 2001. Interaction of scorpion alpha-toxins with cardiac sodium channels: binding properties and enhancement of slow inactivation. Journal of General Physiology 117 (6), 505–518. Chen, L.Q., Santarelli, V., Horn, R., Kallen, R.G., 1996. A unique role for the S4 segment of domain 4 in the inactivation of sodium channels. Journal of General Physiology 108 (6), 549–556. Chen, H., Gordon, D., Heinemann, S.H., 2000. Modulation of cloned skeletal muscle sodium channels by the scorpion toxins Lqh II, Lqh III, and Lqh alphaIT. European Journal of Physiology 439 (4), 423–432. Chen, B., Wang, C., Ji, Y., 2001Scorpion BmK venom induces nociceptive response of rats by plantar injection. Neurotoxicology and Teratology 23 (6), 675– 679. Chen, H., Lu, S., Leipold, E., Gordon, D., Hansel, A., Heinemann, S.H., 2002. Differential sensitivity of sodium channels from the central and peripheral nervous system to the scorpion toxins Lqh-2 and Lqh-3. European Journal of Neuroscience 16 (4), 767–770. Chen, J., Tan, Z.Y., Zhao, R., Feng, X.H., Shi, J., Ji, Y.H., tul=0?>Chen et al., 2005. The modulation effects of BmK I, an alpha-like scorpion neurotoxin, on voltagegated Na+ currents in rat dorsal root ganglion neurons. Neuroscience Letters 390 (2), 66–71. Clancy, C.E., Rudy, Y., 2002. Na+ channel mutation that causes both Brugada and long-QT syndrome phenotypes: a simulation study of mechanism. Circulation 105 (10), 1208–1213. Dumaine, R., Wang, Q., Keating, M.T., Hartmann, H.A., Schwartz, P.J., Brown, A.M., Kirsch, G.E., 1996. Multiple mechanisms of Na+ channel-linked long-QT syndrome. Circulation Research 78 (5), 916–924. Eaholtz, G., Scheuer, T., Catterall, W.A., 1994. Restoration of inactivation and block of open sodium channels by an inactivation gate peptide. Neuron 12 (5), 1041– 1048. Eaholtz, G., Zagotta, W.N., Catterall, W.A., 1998. Kinetic analysis of block of open sodium channels by a peptide containing the isoleucine, phenylalanine, and methionine (IFM) motif from the inactivation gate. Journal of General Physiology 111 (1), 75–82. Gellens, M.E., George Jr., A.L., Chen, L.Q., Chahine, M., Horn, R., Barchi, R.L., Kallen, R.G., 1992. Primary structure and functional expression of the human cardiac tetrodotoxin-insensitive voltage-dependent sodium channel. Proceedings of National Academy of Science 89 (2), 554–558. Gilles, N., Blanchet, C., Shichor, I., Zaninetti, M., Lotan, I., Bertrand, D., Gordon, D., 1999. A scorpion alpha-like toxin that is active on insects and mammals reveals an unexpected specificity and distribution of sodium channel subtypes in rat brain neurons. Journal of Neuroscience 19 (20), 8730–8739.
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Goudet, C., Huys, I., Clynen, E., Schoofs, L., Wang, D.C., Waelkens, E., Tytgat, J., 2001. Electrophysiological characterization of BmK M1, an alpha-like toxin from Buthus martensi Karsch venom. FEBS Letters 495 (1–2), 61–65. Han, C., Tavi, P., Weckstrom, M., 2002. Role of the Na+–Ca2+ exchanger as an alternative trigger of CICR in mammalian cardiac myocytes. Biophysical Journal 82 (3), 1483–1496. Ji, Y.H., Mansuelle, P., Terakawa, S., Kopeyan, C., Yanaihara, N., Hsu, K., Rochat, H., 1996. Two neurotoxins (BmK I and BmK II) from the venom of the scorpion Buthus martensi Karsch: purification, amino acid sequences and assessment of specific activity. Toxicon 34 (9), 987–1001. Jover, E., Martin-Moutot, N., Couraud, F., Rochat, H., 1980. Binding of scorpion toxins to rat brain synaptosomal fraction. Effects of membrane potential, ions, and other neurotoxins. Biochemistry 19 (3), 463–467. Kontis, K.J., Goldin, A.L., 1997. Sodium channel inactivation is altered by substitution of voltage sensor positive charges. Journal of General Physiology 110 (4), 403–413. Leipold, E., Lu, S., Gordon, D., Hansel, A., Heinemann, S.H., 2004. Combinatorial interaction of scorpion toxins Lqh-2, Lqh-3, and LqhalphaIT with sodium channel receptor sites-3. Molecular Pharmacology 65 (3), 685–691. Litwin, S.E., Li, J., Bridge, J.H., 1998. Na–Ca exchange and the trigger for sarcoplasmic reticulum Ca release: studies in adult rabbit ventricular myocytes. Biophysical Journal 75 (1), 359–371. Martens, J.R., Navarro-Polanco, R., Coppock, E.A., Nishiyama, A., Parshley, L., Grobaski, T.D., Tamkun, M.M., 2000. Differential targeting of Shaker-like potassium channels to lipid rafts. Journal of Biological Chemistry 275 (11), 7443–7446. Martens, J.R., Sakamoto, N., Sullivan, S.A., Grobaski, T.D., Tamkun, M.M., 2001. Isoform-specific localization of voltage-gated K+ channels to distinct lipid raft populations. Targeting of Kv1.5 to caveolae. Journal of Biological Chemistry 276 (11), 8409–8414. Moran, O., Nizzari, M., Conti, F., 2000. Endogenous expression of the beta1A sodium channel subunit in HEK-293 cells. FEBS Letters 473 (2), 132–134. Mozhayeva, G.N., Naumov, A.P., Nosyreva, E.D., Grishin, E.V., 1980. Potentialdependent interaction of toxin from venom of the scorpion Buthus eupeus with sodium channels in myelinated fibre: voltage clamp experiments. Biochimica et Biophysica Acta 597 (3), 587–602. Noble, D., Noble, P.J., 2006. Late sodium current in the pathophysiology of cardiovascular disease: consequences of sodium–calcium overload. Heart 92 (Suppl. 4), iv1–iv5. Possani, L.D., Becerril, B., Delepierre, M., Tytgat, J., 1999. Scorpion toxins specific for Na+-channels. European Journal of Biochemistry 264 (2), 287–300. Rogers, J.C., Qu, Y., Tanada, T.N., Scheuer, T., Catterall, W.A., 1996. Molecular determinants of high affinity binding of alpha-scorpion toxin and sea anemone toxin in the S3–S4 extracellular loop in domain IV of the Na+ channel alpha subunit. Journal of Biological Chemistry 271 (27), 15950–15962. Sautiere, P., Cestele, S., Kopeyan, C., Martinage, A., Drobecq, H., Doljansky, Y., Gordon, D., 1998. New toxins acting on sodium channels from the scorpion Leiurus quinquestriatus hebraeus suggest a clue to mammalian vs insect selectivity. Toxicon 36 (8), 1141–1154. Sheets, M.F., Hanck, D.A., 1995. Voltage-dependent open-state inactivation of cardiac sodium channels: gating current studies with Anthopleurin-A toxin. Journal of General Physiology 106 (4), 617–640. Sun, H.Y., Zhu, H.F., Ji, Y.H., n et al., 2003. BmK I, an alpha-like scorpion neurotoxin, specifically modulates isolated rat cardiac mechanical and electrical activity. Sheng Li Xue Bao 55 (5), 530–534. Sun, H.Y., Zhou, Z.N., Ji, Y.H., n et al., 2005. The role of voltage-gated Na+ channels in excitation-contraction coupling of rat heart determined by BmK I, an alpha-like scorpion neurotoxin. Toxicology in Vitro 19 (2), 183–190. Veldkamp, M.W., Viswanathan, P.C., Bezzina, C., Baartscheer, A., Wilde, A.A., Balser, J.R., 2000. Two distinct congenital arrhythmias evoked by a multidysfunctional Na+ channel. Circulation Research 86 (9), E91–E97. Wang, S.Y., Wang, G.K., 2003. Voltage-gated sodium channels as primary targets of diverse lipid-soluble neurotoxins. Cell Signalling 15 (2), 151–159. Wang, Q., Shen, J., Splawski, I., Atkinson, D., Li, Z., Robinson, J.L., Moss, A.J., Towbin, J.A., Keating, M.T., 1995. SCN5A mutations associated with an inherited cardiac arrhythmia, long QT syndrome. Cell 80 (5), 805–811. Yu, F.H., Catterall, W.A., 2003. Overview of the voltage-gated sodium channel family. Genome Biology 4 (3), 207. Zlotkin, E., 1999. The insect voltage-gated sodium channel as target of insecticides. Annual Review of Entomology 44, 429–455.
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Electrophysiological characterization of BmK I, an a-like scorpion toxin, on rNav1.5 expressed in HEK293t cells Xing-Hua Feng a, Jue-Xu Chen b, Ying Liu a, Yong-Hua Ji b,* a b
Graduate School of the Chinese Academy of Sciences, Shanghai Institute of Physiology, Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, PR China School of Life Sciences of Shanghai University, Shang-Da Road 99, Shanghai 200444, PR China
a r t i c l e
i n f o
Article history: Received 30 March 2008 Accepted 23 June 2008 Available online 28 June 2008 Keywords: HEK293t cell Cardiac sodium channels BmK I Patch clamp
a b s t r a c t A recent study described the pharmacological properties of BmK I, an a-like toxin from the Chinese scorpion Buthus martensi Karsch, on the cardiac sodium channel (hH1) expressed in Xenopus oocytes. Considering that a-like toxins are unique in their inability to bind to rat synaptosomes despite a high toxicity by intravenous injection, the present study investigated the pharmacological properties of BmK I on rNav1.5 expressed in a mammalian HEK293t cell line. The results include: (1) BmK I slowed and partially inhibited the inactivation of rNav1.5, produced a substantial persistent current and increased peak current (the EC50 for increasing peak current by BmK I was 99.4 ± 20.1 nM); (2) BmK I delayed the recovery of the sodium channel from inactivation; (3) after exposure to 300 nM BmK I, the steady-state activation curve of rNav1.5 was negatively shifted by about 19 mV; and (4) the association of BmK I and rNav1.5 was faster than their dissociation. The results show that BmK I displayed the pharmacological characteristics of an a-like toxin on rNav1.5 channels expressed in HEK293t cells, and suggested that the host expression system should be taken into consideration when characterizing the pharmacological properties of toxins. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Voltage-gated sodium channels (VGSCs) respond to the depolarizing drive associated with the generation of action potentials in most excitable cells. In vivo, VGSCs usually consist of a large pore-forming a-subunit and one or more auxiliary subunits. The a-subunit has a highly conserved structure composed of four homologous domains (DI-DIV), each with six transmembrane segments (S1–S6) and a hairpin-like pore region between S5 and S6. Due to the structural conservation and their functional importance in cellular excitability (Yu and Catterall, 2003; Zlotkin, 1999), VGSCs are targeted by a variety of toxins, including peptides from scorpion venom (Wang and Wang, 2003). Most scorpion neurotoxins targeting VGSCs are single chain polypeptides composed of 60–70 amino acids cross-linked by four disulfide bridges (Arnon et al., 2005; Chen et al., 2002; Possani et al., 1999). These neurotoxic polypeptides comprise two main groups: a- and b-toxins (Gordon et al., 1996; Leipold et al., 2004). Scorpion
Abbreviations: VGSCs, voltage-gated sodium channels; BmK, Buthus martensi Karsch; EC50, half maximal effective concentration; HEK, human embryonic kidney; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; BSA, bovine serum albumin; HEPES, (N-(2-hydroxyethyl) piperazine-N0 -(2-ethanesulfonic acid)). * Corresponding author. Tel./fax: +86 21 66135189. E-mail addresses: [email protected], [email protected], [email protected] (Y.-H. Ji). 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.06.009
a-toxins bind to site 3, which is occupied by several groups of polypeptide: a-scorpion toxins, sea-anemone toxins and some spider toxins. Scorpion a-toxins prolong the action potential by slowing the inactivation of VGSCs. According to the different pharmacological and binding properties, the a-toxins can be further divided into three subgroups: classical a-, a-like, and insect a-toxins. Classical a-toxins are highly toxic to mammals, whereas the insect a-toxins are highly toxic to insects. The a-like toxins act on both mammals and insects, but they are unique in their inability to bind to rat synaptosomes despite a high toxicity by intravenous injection (Cestele et al., 1999; Gilles et al., 1999; Sautiere et al., 1998). BmK I is an a-like toxin from the eastern Asian scorpion Buthus martensi Karsch (Ji et al., 1996). BmK I acts on VGSCs in the central and peripheral nervous systems, thus producing epileptic seizures and hyperalgesia (Bai et al., 2006; Chen et al., 2001). Interestingly, BmK I also functions as a cardiotoxin: it increases rat heart contractility and prolongs inactivation of sodium currents in isolated rat ventricular myocytes (Sun et al., 2003; Sun et al., 2005). Since Nav1.5 is the predominant isoform expressed in heart muscle (Gellens et al., 1992), BmK I was postulated to be active on this cardiac sodium channel isoform. Consistent with this speculation, the inactivation of hNav1.5 expressed in Xenopus oocytes could be slowed down by BmK I (Goudet et al., 2001). It has been reported, however, that VGSCs expressed in Xenopus oocytes exhibit different electrophysiological properties (e.g., the slow mode of gating) compared to in vivo or when mammalian cells are used as expression systems. These differences might
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further influence pharmacological effects of toxins targeting VGSCs. Therefore, in the present paper, we studied the pharmacological effects of the presumptive cardiotoxin on a subunit of cardiac sodium channel isoform-rNav1.5, expressed in a mammalian HEK293t cell line. 2. Material and methods 2.1. Material BmK venom collected by electric stimulation was purchased from a scorpion farm in Zhengzhou, Henan province, China. BmK I was purified according to the method described by Ji et al. (1996). The purity of this component was confirmed by means of either mass spectrometry or sequencing determination. HEK293T cell line was from Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Unless otherwise stated, all chemicals were from Sigma. 2.2. Cell culture and transient transfection HEK293t cells were maintained in DMEM (GIBCO BRL) supplemented with 10% FBS, 20 lg/ml penicillin, and 10 lg/ml streptomycin in a 5% CO2 incubator. The cell line was transfected with plasmids coding a subunit of rat cardiac sodium channel (rNav1.5) by means of Lipofectamine2000 (Invitrogen) reagent according to the instructions of the manufacturer. Briefly, HEK293t cells were passaged onto 35 mm culture plates at about 25% confluence. Following incubation for 24 h, cells were exposed to a 2 ml Opti-MEM medium (GIBCO Life Technologies) containing 2 ll Lipofectamine2000 and 1–2 lg sodium channel expression vector. Following a 5–24 h incubation at 37 °C with humidified 5% CO2, the medium was exchanged for normal HEK293t cell growing medium. A post-transfection incubation for 36–48 h preceded the electrophysiological recordings. Cells with inward currents were deemed as transfection-positive ones. Whole-cell patch clamp recordings were conducted at room temperature using an Axopatch-200B amplifier (Axon Instruments Inc., Union, CA). Electrophysiological recordings were started 10 min after establishing a whole-cell configuration. To eliminate the effect of steady-state inactivation during experiments, the holding potential was set to 140 mV. The series resistance was compensated (70–80%) to minimize voltage errors. Leak subtraction was performed using a P/4 protocol applied before the test protocol for all voltage clamp recordings. Signals were low-pass filtered at 5 kHz and digitized at 20 kHz. We used patch pipettes that had a resistance of 2–3 MX when filled with the pipette solution listed below. The pipette solution contained (mM): CsF 140, CsCl 10, MgCl2 1, EGTA 2, HEPES 10 (pH 7.3). The bath solution contained (mM): NaCl 140, MgCl2 1, CaCl2 2, HEPES 10, Glucose 10 (pH 7.3). 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 BSA alone did not alter sodium 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 lm from the cells. After 10-min perfusion with the toxin, step pulses as detailed in the figure legends were used to investigate the effect of BmK I on rNav1.5 channels. 2.3. Data analysis Conductance was calculated as G(V) = Ipeak(V)/(V Erev), where the reversal potential (Erev) was measured experimentally for each cell.
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The voltage dependence of activation was quantified by fitting the conductance measures to a Boltzmann function: G(V) = Gmax/ [1 + exp (Vtest V1/2)/K]. Steady-state inactivations were fitted to a Boltzmann function with a non-zero pedestal (I0) calculated as I/Imax = (1 I0)/ [1 + exp (V1/2 Vtest)/K] + I0, where V1/2 was the test potential at which the channels were half-maximally activated and K was the slope factor. The data in Figs. 1B, 2 and 4C were fitted with a single exponential function: y = A exp (t/s) + y0, where s was the time constant. The equation used for fitting dose–response relationships was Inorm = A/[1 + ([BmK I]/EC50)p] + C, where Inorm was the measured and normalized peak current, EC50 was the half maximal effective concentration, and p was the slope factor. Data were analyzed using the Clampfit8.2 (Axon Instruments, USA) and Origin6.0 (OriginLab Corp., Northampton, MA, USA), and they were presented as means ± SEM. The number of cells examined was represented by n. Student’s paired or unpaired t-tests were used for comparisons, and statistical significance was assessed as P < 0.05.
3. Results 3.1. Effects of BmK I on rNav1.5 sodium currents Representative sodium currents recorded from a HEK293t cell expressing rNav1.5 in the absence and in the presence of 300 nM BmK I were shown in Fig. 1A, which shows that BmK I strongly slowed down the inactivation of sodium currents and caused persistent currents. Under control conditions, inactivation increased with increasing voltage, which was maximal at around 10 mV. After application of 300 nM BmK I, inactivation was strongly impaired. Comparison of time constants for inactivation in control and in the presence of BmK I shows that inactivation was very significantly slowed over potentials from 40 to 0 mV (Fig. 1B). The relative persistent currents were obtained by dividing corresponding peak currents (Ipeak) by persistent currents (Ipersistent). In the absence of BmK I, there were only very small relative persistent currents; in the presence of 300 nM BmK I, relative persistent currents increased with increasing depolarizations. Comparison with control shows that BmK I significantly induced persistent currents at potentials more positive than 30 mV (Fig. 1C). The corresponding I–V curves were shown in Fig. 1D. It indicates that BmK I significantly increased the peak current over the range from 50 to +10 mV. Moreover, this effect of BmK I on peak currents was dose-dependent (Fig. 1E). After 10-min treatment with different concentration of BmK I, currents were elicited by 70-ms depolarizations to 30 mV from the holding potential of 140 mV. The increase for relative increment peak current enhanced from 3% by 30 nM BmK I (n = 5) to 18% after exposure to 100 nM BmK I (n = 5). The relative increment was approximately 46% and 43% in the presence of 300 nM (n = 4) and 1000 nM (n = 5) BmK I, respectively. The EC50 value determined by a sigmoidal fit of the data in Fig. 1E was 99.4 ± 20.1 nM. 3.2. BmK I slowed the recovery of rNav1.5 channels Because BmK I impaired the inactivation of rNav1.5 channels, we further investigated its effect on the recovery from inactivation (Fig. 2). In the absence and presence of 300 nM BmK I, recovery increased with intervals at 140 mV. Under control conditions, the recovery of rNav1.5 was fast, with the time constant of 2.31 ± 0.32 ms. After treatment with 300 nM BmK I, the recovery
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Fig. 1. Functional effect of BmK I on rNav1.5 currents. (A) Current responses to test pulses for rNav1.5 channels under control conditions (left) and after application of 300 nM BmK I (right). 70-ms test pulses ranged from 100 mV to +40 mV by a 10-mV increment from the holding potential of 140 mV. For clarity, only the first 40 ms of the currents are shown. (B) Effect of 300 nM BmK I on the relative persistent currents, here expressed as Ipersistent (% Ipeak). Persistent currents (Ipersitent) were measured as the mean current remaining between 45 and 65 ms after the start of depolarizations. (C) Current–voltage (I–V) curves of sodium currents in the absence (closed squares) and presence of 300 nM BmK I (closed circles). (n = 6); *P < 0.05. (D) Time constants for inactivation (Tau) in the absence (closed squares) and presence of 300 nM BmK I (closed squares). The time constants were obtained by fitting the declining phase of sodium currents with single exponential functions. The holding potential was 140 mV. n = 5; ** P < 0.01. (E) Dose-dependent enhancement of the peak rNav1.5 current induced by BmK I. After 10-min treatment with different concentration of BmK I, currents were elicited by 70-ms depolarizations to 30 mV from the holding potential of 140 mV. Relative increments of peak currents were calculated by dividing the increments induced by BmK I with the peak current under control, and plotted against the concentration of BmK I. The EC50 value determined by a sigmoidal fit is 99.4 ± 20.1 nM.
was significantly slowed down, with the time constant of 5.36 ± 0.51 ms. In addition, 300 nM BmK I decreased the percentage of available sodium channels after 30-ms recovery at 140 mV. The fraction recovery of rNav1.5 dropped from 93% (control) to 84% (300 nM BmK I). This indicates that, in the presence of BmK I, more sodium channels were in an inactivation state, requiring more time to recover. 3.3. BmK I enhanced the voltage-dependent activation of rNav1.5 channels BmK I could induce rNav1.5 currents at more negative test voltage, which can be deduced from Fig. 1D. This caused a dose-depen-
dent, hyperpolarizing shift in the steady-state activation curve (Fig. 3A). Under control conditions, the half-activation voltage (V1/2) of rNav1.5 was 36.85 ± 1.43 mV. In the presence of 30 nM BmK I (n = 4), the shift of V1/2 was 5.06 ± 1.64 mV. The shift by 100 nM (n = 7) and 300 nM (n = 5) BmK I was 10.22 ± 1.22 mV and 18.78 ± 2.45 mV, respectively. There was, however, no significant difference between slopes of these curves. To study steady-state inactivation, cells were held for 400 ms at prepulse potentials ranging from 140 to +20 mV and then subjected to a depolarizing pulse of 30 mV for 40 ms. The steady-state inactivation curves in the absence and presence of BmK I were displayed in Fig. 3B. Statistical analyses show that there was no significant effect of BmK I on steady-state inactivation.
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Fig. 2. Effect of BmK I on the recovery kinetics of rNav1.5 channels. To measure recovery from inactivation, a 30-ms conditioning step to 30 mV was applied and then availability was assayed with a test step to 30 mV after variable recovery intervals at 140 mV. Each step pair was delivered at a frequency of 0.2 Hz. Fraction of available channels was calculated as the ratio of the test to the conditioning current amplitude and was plotted versus recovery interval in absence (closed squares) and presence of 300 nM BmK I (closed circles). Data were fitted with single exponentials. Control: n = 9; 300 nM BmK I: n = 6. *P < 0.05.
3.4. Kinetics of BmK I acting on rNav1.5 channels To investigate the kinetics of BmK I acting on rNav1.5 channels, the association and disassociation rat of BmK I and rNav1.5 channels was studied. Representative traces for association were shown in Fig. 4A, and those for dissociation in Fig. 4B. Comparison of the time constants from Fig. 4C shows that the binding process of BmK I, with time constant of 21.48 ± 1.68 s was much faster than that of unbinding, with time constant of 73.09 ± 17.19 s. Two traces shown in Fig. 4D were the first current in Fig. 4A (before application of 300 nM BmK I) and the last one in Fig. 4B (after a 10-min washout), respectively. There was nearly no difference between them, which suggests that the effect of BmK I on rNav1.5 was almost completely reversible within the time span of the experiment. 4. Discussion In this study, BmK I was demonstrated to slow down the inactivation of sodium currents and induce persistent currents, which indicates that BmK I is active on the rNav1.5 transiently expressed in HEK293t, reinforcing the previous conclusions that BmK I is an a-like toxin and that cardiac sodium channels are one of the targets of BmK I. The intracellular linker between DIII and DIV, in which the critical motif is the hydrophobic triad IFM, and the S4 in DIV, which is in juxtaposition with the receptor site of a-like scorpion toxins (DIV S3–S4) (Rogers et al., 1996), are both involved in the fast inactivation of sodium channels (Chen et al., 1996; Eaholtz et al., 1994; Eaholtz et al., 1998; Kontis and Goldin, 1997). Thus, it was believed that the binding of a-like scorpion toxins (including BmK I) hampered the movement of DIV S4 (Sheets and Hanck, 1995) required for the formation of binding site for IFM, resulting in the slowing the formation of receptor site for IFM, so the inactivation was slowed down. Moreover, in the presence of BmK I, the receptor site for IFM formed in response to depolarizations had less affinity, which leaded to the equilibrium between bound and unbound states more partially favoring the unbound state, which resulted in persistent currents. BmK I delayed the recovery of rNav1.5 channels from inactivation. Moreover, our study indicates that, in the presence of BmK I,
Fig. 3. Effects of BmK I on the gating of the rNav1.5 channel. (A) Effect of BmK I on the steady-state activation of rNav1.5. Steady-state activation was studied by measuring the peak sodium conductance during a 70-ms test pulse to various test potentials, in 10-mV increment, from a 140 mV holding voltage. Normalized conductance (expressed as the conductance relative to the maximum conductance G/Gmax) was plotted against test voltages and fitted with Boltzmann functions. The lines showed the fit of the means of control (solid line), 10 (dashed line), 100 (dotted line) and 300 nM (dash dot line) BmK I (n = 5), respectively. (B) Effect of BmK I on the steady-state inactivation of rNav1.5. Steady-state inactivation curves were determined using 400-ms conditioning pulses to voltages between 140 and +20 mV and a standard 40-ms test pulse to 30 mV. Normalized test currents were plotted against the conditioning voltages and fitted with Boltzmann functions. The lines showed the fit of the means of control (solid line), 10 (dashed line), 100 (dotted line) and 300 nM (dash dot line) BmK I, respectively. Control: n = 5; 10 nM BmK I: n = 4; 100 nM BmK I: n = 7; 300 nM BmK I: n = 5.
more sodium channels were in another inactivation state (84% vs. 93% channel available after 30-ms recovery at 140 mV), which needed more time to recover. As such, it was assumed that, under control conditions, the recovery from fast inactivation was dominant. In the presence of BmK I, after stimulation, more sodium channels were in the inactivation state requiring more recovery time, and this made a significant contribution to total recovery. Thus, the recovery was slowed down. This effect of BmK I was in accordance with result obtained by Chen and coworkers, who demonstrated that an a-like toxin, Lqh III, slowed down the recovery rate from inactivation of cardiac sodium channel hH1 (Chen and Heinemann, 2001). BmK I induced a shift of the steady-state activation curve to more negative potentials, whereas voltage dependence of steady-
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Fig. 4. Association and disassociation kinetics of BmK I on rNav1.5 channels. (A) Representative recording of association obtained during exposure to 300 nM BmK I. (B) Representative recordings of dissociation during washout with bath solution. Representative currents were elicited with ten 90-ms pulses applied from 140 mV to 30 mV every 30 seconds. The first trace of association (dissociation) was recorded immediately once applying 300 nM BmK I (bath solution). The holding potential was 140 mV. The vertical lines indicate 10 ms after the test pulses start. (C) The association and disassociation rate of BmK I and rNav1.5. The association and dissociation rate were estimated by the normalized sodium currents at 10 ms after the test pulse start (I10 ms/Imax at 10 ms). Normalized sodium currents of association (closed squares) and dissociation (closed circles) were plotted against the application time and fitted by single exponential functions. The time constants of association and dissociation were estimated to be 21.48 ± 1.68 s and 73.09 ± 17.19 s, respectively. (D) Two superimposed currents. The current with mark ‘‘control” was the first trace in Fig. 4A and that with ‘‘wash” was the last one in Fig. 4B (n = 5).
state inactivation was not affected. This finding was in agreement with that obtained on TTX-sensitive sodium currents recording from DRGs (Chen et al., 2005) and with findings from research of LqhIII on skeletal muscle sodium channels expressed in HEK293t cells (Chen et al., 2000). These results might imply a common mode of action for a-like scorpion toxins on the gating properties of sodium channels in vivo resulting probably from a common binding site for these polypeptide neurotoxins, which warranted further investigation. It is noteworthy that, in contrast to our results, Goudet et al. reported that BmK I induced a hyperpolarizing shift of the steady-state inactivation of the human cardiac sodium channels expressed in Xenopus oocytes (Goudet et al., 2001). This discrepancy was postulated to result from the different host cells. It was highly suspected that the auxiliary b1-subunit, stably expressed by HEK293t cells (Moran et al., 2000) but not in oocytes, and the different lipid content (Martens et al., 2000, 2001) might be involved in the discrepancy. Other possibilities cannot, however, be ruled out. Our work shows that association of BmK I to cardiac sodium channels was more rapid than dissociation. Moreover, in our study, it was observed that the effect of BmK I was only partially reversible (data not shown), which was also reported in DRGs (Chen et al., 2005). The result in Fig. 4D, however, seems to show that the effect of BmK I could be almost completely reversible. Since site 3 toxins bind with sodium channels in a voltage-dependent manner and depolarization induced a dramatic decrease of a-scorpion toxins affinity to sodium channels (Chen et al., 2000; Jover et al., 1980; Mozhayeva et al., 1980), this discrepancy should be the result of the repetitive long test pulses during washout, which is also indirect evidence for voltage-dependent binding of BmK I to rNav1.5.
Nav1.5 was the predominant sodium channel subtype in heart muscles (Gellens et al., 1992), and played a key role in the cardiac excitability and conduction. Since the important role of normal Nav1.5 function in cardiac rhythm had been highlighted by studies characterizing the mutant resulting in LQT3 and Brugada syndrome (Dumaine et al., 1996; Veldkamp et al., 2000; Wang et al., 1995), it was suggested that BmK I-induced inactivation-slowing and persistent current were at least partly responsible for cardiac arrhythmia induced by exposure of isolated rat hearts to BmK I (Sun et al., 2003). Moreover, an increase in the amplitude and voltage range of the so-called sodium ‘‘window current”; i.e., the maintained inward sodium current caused by an overlap in activation and inactivation relations, resulting from the negative shift of voltage-dependent activation, might also partially account for cardiac arrhythmia induced by BmK I. Furthermore, as a consequence of inactivation impairment, persistent current and a larger window current in the presence of BmK I, sodium channels could conduct much more inward sodium current than in the absence of the toxin. The sodium gradient becomes insufficient to maintain the calcium gradient, so intracellular calcium increases through sodium–calcium exchange (Han et al., 2002; Litwin et al., 1998; Noble and Noble, 2006). This might explain the increase in rat heart contractility induced by BmK I. Finally, the slower recovery from inactivation decreases the density of available cardiac sodium channels during the cardiac cycle, which is thought to be one cause of arrhythmogenesis in patients with Brugada and Long-QT syndrome (Clancy and Rudy, 2002). In conclusion, the present study shows that the scorpion toxin BmK I exhibited a-like toxin properties on rNav1.5 expressed in HEK293t cells. BmK I impaired inactivation processes and induced substantial persistent sodium currents. Moreover, BmK I slowed
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