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The role of voltage-gated Na+ channels in excitation–contraction coupling of rat heart determined by BmK I, an α-like scorpion neurotoxin
Toxicology in Vitro 19 (2005) 183–190 www.elsevier.com/locate/toxinvit
The role of voltage-gated Na+ channels in excitation–contraction coupling of rat heart determined by BmK I, an a-like scorpion neurotoxin Hai-Ying Sun a
a,b
, Zhao-Nian Zhou a, Yong-Hua Ji
a,c,*
The Key Laboratory of Neurobiology, Institute of Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, PR China b School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200030, PR China c School of Life Sciences, Shanghai University, Shanghai 200436, PR China Received 31 December 2003; accepted 7 July 2004
Abstract A mechanism underlying the increase in rat heart contractility modulated by BmK I, an a-like scorpion neurotoxin, was investigated using whole-cell patch-clamp and fluorescence digital imaging techniques. Results showed that (a) L-type Ca2+ current could not be modified by 500 nM BmK I; (b) The inactivation process of Na+ current was significantly delayed with no change of its amplitude; (c) The overall intracellular Na+ and Ca2+ concentration could be augmented in the presence of BmK I; (p < 0.05); (d) The increase of free intracellular Ca2+ concentration induced by BmK I was inhibited completely by 5 mM NiCl2 (p < 0.05), an inhibitor of Na+–Ca2+ exchanger; (e) The spontaneous Ca2+ release induced by 10 mM caffeine from sarcoplasmic reticulum could not be modulated by 500 nM BmK I in the absence of external Ca2+. These results indicate that cardiac voltage-gated Na+ channels are also targets of BmK I. Na+ accumulation through Na+ channels can trigger sarcoplasmic reticulum Ca2+ release in rat cardiac myocytes via reverse-mode Na+–Ca2+ exchanger. Furthermore, Ca2+ release from sarcoplasmic reticulum induced by BmK I most likely involves a Ca2+-induced release mechanism. 2004 Elsevier Ltd. All rights reserved. Keywords: Inotropism; Voltage-gated Na+ channel; Reverse-mode Na+–Ca2+ exchanger; BmK I
Voltage-gated Na+ channels (VGSCs) composed of pore-forming a and b subunits are responsible for the generation and propagation of action potential in excitable cells (Catterall, 2000). Nine genes encoding a subunits have been functionally expressed. These isoforms can be differentiated by their distinct patterns of development and localization in the nervous system, skeletal and cardiac muscle, as well as the relative sensitivity to tetrodotoxin (TTX) (Gellens et al., 1992; Goldin et al., 2000). Isoforms expressed in the central nervous system *
Corresponding author. Tel.: +86 21 54920300; fax: +86 21 54920276. E-mail address: [email protected] (Y.-H. Ji). 0887-2333/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2004.07.005
(Nav1.1, 1.2, 1.3 and 1.6) are inhibited by nanomolar concentrations of TTX, as is the isoform present in adult skeletal muscle (Nav1.4). In contrast, the primary cardiac isoform (Nav1.5) requires micromolar concentrations of TTX for inhibition (Goldin, 2001). Recently, VGSCsmediated cardiac calcium-induced calcium release (CICR) and contraction have been extensively investigated in cat and guinea pig ventricular cells. However, the results obtained from different experimental protocols seem to be controversial (Levi et al., 1993; Renaud et al., 1986; Sham et al., 1992; Lipp and Niggli, 1994). Thus, the study on how VGSCs (Nav1.5) modulate heart contractility is worthwhile to pursue to understand excitation–contraction (E–C) coupling in rat heart.
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It is well known that VGSCs are the targets of numerous natural neurotoxins, which have been extensively examined to approach to the gating mechanism of VGSCs involved in neuronal signaling transmission such as algesia and analgesia (Josephine et al., 2002; Decosterd et al., 2002; Tyrrell et al., 2001; Benn et al., 2001; Gold, 1999; Cestele and Catterall, 2000; Denac et al., 2000; Tan et al., 2001). Most long-chain neurotoxic polypeptides purified from the scorpion venoms are also determined to be VGSCs-specific modulators. Generally, they are classified into two main classes, the a- and b-toxins according to their modulating effects on Na+ currents and binding characteristics to VGSCs (Gordon et al., 1998). Among them, BmK I was one of the most toxic component purified from the venom of Chinese scorpion Buthus martensi Karsch (BmK) with nociceptive-inducing activity (Ji et al., 1996; Chen et al., 2001). BmK I could favorably modulate the inactivation phase of VGSCs in crayfish axons in the same mode as classic a-toxins (Terakawa et al., 1989), and also could interact with insect VGSCs that distinguished it from classical a-toxins such as AaH II (Gordon et al., 1996). It is surprising that BmK I was unable to compete with the binding of either a- or b-toxins to receptor site 3 and 4 of mammal brain-type Na+ channels. Therefore, it is considered to belong to the a-like group (Cestele et al., 1999; Vargas et al., 1987; Li and Ji, 2000). Although the modulating activity of a few neurotoxins on either guinea pig or rat heart contractile force and the Na+ currents have been demonstrated, such as TsTX and TiTXc from Brazilian scorpion Tityus serrulatus, and AxII from Anthopleura xanthogrammica (Almeida et al., 1982; Marcotte et al., 1997; Renaud et al., 1986; Goudet et al., 2001), it remains to be unraveled whether a-like scorpion neurotoxin BmK I is sensitive to rat cardiac VGSCs, and how VGSCs are involved in signaling pathway of rat cardiac excitation–contraction (E–C) coupling so far. Our previous study indicated that BmK I could increase rat heart contractility in a dose-dependent manner (Sun et al., 2003). The present study was designed to investigate the modulating effects of BmK I on VGSCs in rat cardiac myocytes, and to determine the potential mechanisms underlying the increase of rat cardiac contractility. Furthermore, it might provide insight into the receptor sites-specific association with BmK I between neuronal and cardiac VGSCs.
moved and perfused retrogradely through the aorta with Ca2+-free TyrodeÕs solution at 37 C, followed by the Ca2+-free TyrodeÕs solution containing 50 lM Ca2+, 1% BSA and 0.04% type II collagenase (Sigma, USA) for 5–10 min. After perfusion, the separated ventricles were chopped into small pieces, incubated in fresh TyrodeÕs solution for 10 min. The isolated cells were filtrated through a nylon mesh (200 lm) and suspended in the TyrodeÕs solution, in which the Ca2+ concentration gradually increased to 1.0 mM. Only the cells showed rod shaped and clear cross striation were used for the experiments. The Ca2+-free TyrodeÕs solution contained: NaCl (135 mM), KCl (5.4 mM), MgCl2 (1.0 mM), NaH2PO4 (0.33 mM), HEPES (5.0 mM), and Glucose (10 mM) adjusted to pH 7.4 with 0.5 N NaOH. 1.2. Recording of INa and ICa The whole-cell patch clamp technique was employed to record INa and ICa in single ventricular myocyte. All recordings were carried out at room temperature (20– 22 C). The cells were constantly perfused after adhering to the bottom of the chamber. Membrane currents were recorded with EPC9 amplifier (HEKA elektronik, Germany) controlled by EPC9 data acquisition system. The microelectrodes fabricated with microelectrodes puller (PP-830, Narishige, Japan) had a resistance of 2–3 MX when filled with pipette solution. In the whole cell configuration, the series resistance was partially compensated by about 70–80%. Currents were filtered at 5 kHz using a four-pole low-pass Bessel filter. Digital leak subtraction of the current records was carried out by a P/4 protocol. The bath solution of recording INa was composed of (mM): NaCl 90, Choline-Cl 40, CsCl 5.4, HEPES 10, MgCl2 1, NaH2PO4 0.33, CaCl2 1.8, Glucose 10 (pH adjusted with CsOH to 7.4). The pipette solution was composed of (mM): CsCl 20, Cs-fluoride 110, NaCl 5, HEPES 5, EGTA 5, MgCl2 1, Na2-ATP 5 (pH adjusted to 7.2 with CsOH). The bath solution of recording ICa was composed of (mM): Choline-Cl 135, CsCl 5.4, HEPES 10, MgCl2 1.0, NaH2PO4 0.33, CaCl2 1.8, Glucose 10 (pH adjusted with CsOH to 7.4). The pipette solution of recording ICa was composed of (mM): CsCl 20, Cs-aspartate 110, HEPES 5, EGTA 10, MgCl2 1.0, Mg2-ATP 5 (pH adjusted to 7.2 with CsOH). 1.3. Loading of Myocytes with Fura-2/AM and measurement of free [Ca2+]i
1. Materials and methods 1.1. Preparation of adult rat ventricular myocytes Cardiac ventricular myocytes were isolated from Sprague–Dawley adult rats using a standard enzymatic technique (Song et al., 1998). Briefly, rat hearts were re-
Single ventricular myocytes were loaded with 2.5 lM fura-2 acetoxymethyl ester mixed to 0.1% Pluronic F127 (Sigma Chemical Co., USA) in TyrodeÕs solution containing 1.8 mM CaCl2 for 30 min at 37 C. The loaded myocytes were then washed twice with TyrodeÕs solution, and incubated for at least 30 min at room temper-
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ature to completely hydrolyze the dye. The fluorescence intensity of dye-loaded cells was 2–3 times higher than background fluorescence with unloaded cells. The background noise could be automatically corrected and subtracted at each experiment in cool-charge coupled system (C4742-95-12NRB, Hamamatsu Photonics K.K., Japan). The coverslip with the loaded cells was introduced into a transparent perfusion chamber positioned in the light path of an inverted microscope (IX-70, Olympus Optical CO., Tokyo, Japan). Alternating excitation of the fluorescent dye at wavelengths of 340 and 380 nm for fura-2 was performed with an AR-Cation measurement system adapted to the microscope. Emitted light (510 ± 7.5 nm) was detected by cool-charge coupled system. The light signal was recorded and analyzed by software of Aquacosmos 1.2. Additions of agents were made through a MASTERFLEX pumps to ensure optimum performance. Because of the inherent problems with calibration of the fura-2 ratio, data were generally expressed in arbitrary units of fluorescence ratio. The ratio = (F340 F340base)/(F380 F380base). For each experiment, 5–7 groups (cells in different coverslips) data (total cell number ranges from 60 to 100) were summed in every group first, and then each sum was averaged by the cell number. The averaged ratio of each group was normalized, and then the normalized ratios of different groups were summed and averaged by the group number. Moreover, the pseudo color figures of fluorescence ratio before and after administration of BmK I were collected. All experiments were performed at 35 C. 1.4. Loading of myocytes with SBFI/AM and measurement of [Na+]i Single ventricular myocytes were loaded with 10 lM benzofuran isophthalate (SBFI-AM) in the form of its acetoxymethyl ester mixed to 0.1% Pluronic F127 (Sigma Chemical Co., USA) in TyrodeÕs solution containing 1.8 mM CaCl2 for 30 min at 37 C. The rest manipulations were the same as that of the free [Ca2+]i. 1.5. The calibration of SBFI fluorescence The calibration of SBFI fluorescence is performed according to the method described previously (Satoh et al., 1994). Briefly, the cells were loaded with SBFI and then perfused with calibration solutions with various Na+ concentration (0, 5, 10, 30, 50 mM). The calibration solutions were made from appropriate mixtures of high Na+ and high K+ solutions. The high Na+ solution contained (in mM): sodium gluconate 110, NaCl 30, MgCl2 0.6, Na-HEPES 10, EGTA 1, pH was adjusted to 7.1 by adding HCl. The composition of high K+ solution was identical to that of high Na+ solution except complete replacement of Na+ by K+.
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Ten micromoles gramicidin D and 100 lM strophanthidin were added. The cells were incubated with each solution for at least 10 min to allow complete equilibration. [Na+]i = Kd · b · (R Rmin)/(Rmax R). R is the obtained 340/380 nm excitation ratio, Rmin is the ratio at 0 mM [Na+]i, Rmax is the ratio at saturating [Na+]i, Kd is the dissociation constant, and b is the ratio of the excitation efficient of free to Na+ bound SBFI at 380 nm. 1.6. Scorpion toxin The purified toxin BmK I was provided by our laboratory (Ji et al., 1996). 1.7. Statistics All averaged and normalized data were presented as mean ± SD. The statistical significance of the differences in the calculated mean values was evaluated using the StudentÕs t-test. P-value is provided in the text. Statistical significance was assumed when P < 0.05.
2. Results 2.1. Delay of inactivation process of INa modulated by BmK I Sodium currents were evoked by step depolarization of 25 ms ranging from 70 to 0 mV, with increments of 10 mV, from a holding potential of 80 mV. Na+ currents inactivated rapidly after activation in control condition (Fig. 1A left panel, n = 6). In the presence of 500 nM BmK I, the inactivation kinetics were potently slowed down at all tested pulse without the change of peak Na+ currents (Fig. 1A right panel, n = 6), just like scorpion a-toxins (Lee et al., 2000). Current–voltage (I– V) relation was determined using 25-ms depolarizing steps every 10 s from 70 mV to +30 mV with a holding potential (HP) of 80 mV. In order to allow for differences in myocytes size, the magnitude of peak INa currents was normalized to cell capacitance (pA/pF). Cell capacitance of myocytes used in our experiments was 71.33 ± 18.61 pF, the series resistance (Rs) was 5.32 ± 1.06 MX (n = 10) during recording process. Results showed that the density of INa in rat ventricular myocytes was increased slightly from 28.77 ± 3.12 pA/ pF to 30.19 ± 3.89 pA/pF after exposure to 500 nM BmK I (Fig 1B n = 10 from six rats, p > 0.05). On the other hand, calcium current was evoked by a step depolarization to 0 mV during 300 ms from a holding potential of 40 mV. Fig 1C displayed that L-type calcium current at 0 mV could not be modified after the application of 500 nM BmK I (n = 6, p > 0.05). Steady-state activation of INa was evaluated by fitting normalized conductance–voltage values to the Boltzman
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Fig. 1. Electrophysiological recording of rat ventricular myocytes Na+ currents and Ca2+ current modulated by BmK I. (A) Representative tracing of Na+ currents evoked by depolarization ranging from 70 mV to 0 mV by 10 mV steps from a holding potential of 80 mV in the absence (left panel) or presence (right panel) of 500 nM BmK I (n = 6). Inactivation phase of Na+ currents was prolonged significantly by 500 nM BmK I. (B) I–V curve of Na+ currents remained the same in the absence or presence of 500 nM BmK I (n = 6). (C) Representative L-type calcium current evoked by a step depolarization to 0 mV during 300 ms from a holding potential of 40 mV in the absence or presence of 500 nM BmK I indicated by arrow. Ltype calcium current could not be modulated by 500 nM BmK I (n = 6). (D) Averaged and normalized steady-state activation curve of Na+ channel in the absence (j) or presence (d) of 500 nM BmK I. Steady-state activation of Na+ channel could not be modulated by 500 nM BmK I (n = 6). (E) Averaged and normalized curve of steady-state inactivation of Na+ channel was shifted slightly toward negative potential in the presence of 500 nM BmK I (n = 6, p > 0.05). Data stand for mean ± SD.
equation, g/gmax = {1/[1 + exp] (V Vh)/s]} 1. V is the test potential, Vh the midactivation or midinactivation potential, and s the slope factor. The activation curve of INa after addition of 500 nM BmK I was nearly overlapping with that of the control (Fig. 1D, n = 6). In the control, the voltage for half-maximal activation (V1/2) and the slope factor of the activation curve were 40.5 ± 1.0 mV and 5.44 ± 0.6 mV, respectively. In the presence of 500 nM BmK I, V1/2 and slope factor were 41.4 ± 1.1 mV and 4.94 ± 0.6 mV, respectively. Steady-state inactivation of INa was determined with a double-pulse protocol consisting of 300 ms conditioning pulse ranging from 120 to 30 mV, and followed by a constant test pulse of 50 ms duration to 30 mV at a pulsing frequency 0.5 Hz. The amplitude of peak INa during test pulse was normalized to the maximum peak current and plotted as function of the conditioning potential. Data was fitted to the Boltzman equation. In control, Vh and slope factor were 55.03 ± 2.1 mV and
15.09 ± 2.0 mV, respectively. In the presence of 500 nM BmK I, the Vh and slope factor were 58.87 ± 2.3 mV and 16.76 ± 2.3 mV (Fig. 1E, n = 6). Five hundred nanomoles BmK I shifted the I–V relation more hyperpolarized to 3.8 mV at which half of the cardiac Na+ channels were inactivated (n = 6, p > 0.05). In comparison with the time constant of inactivation under control condition, the time course of the inactivation phase s at 30 mV was increased about 10-fold in the presence of 500 nM BmK I from 1.53 ± 0.76 ms to 14.43 ± 2.45 ms (n = 6, p < 0.05). 2.2. Increase in [Na+]i and [Ca2+]i evoked by BmK I Treatment with 1 lM BmK I could cause a gradual rise in [Na+]i from normalized ratio of 1.00–1.08 during a 30-min duration, whereas the normalized ratio did not change under control conditions. The cells incubated with normal external solution only displayed a slight in-
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Fig. 2. Augment of the intracellular free Na+ evoked by BmK I. BmK I was applied at the time indicated by arrow. SBFI fluorescence ratio was proportional to the [Na+]i. (A) Calibrated [Na+]i concentration curve. (B) Intracellular Na+ was increased by BmK I. The number of cells varied from 60 to 100 in each group. *p < 0.05 vs control group 20 min after administration of 1 lM BmK I.
crease of [Na+]i from 1 to 1.005. One micromole BmK I could evoke maximal increase of [Na+]i (Fig. 2B, n = 60– 100, p < 0.05). In order to observe the changes of [Ca2+]i, 50 mM KCl was applied to make a depolarization and trigger CICR. Compared to control, [Ca2+]i was significantly elevated by BmK I in a dose-dependent manner (Fig. 3B, n = 60–100, p < 0.05). Although free [Ca2+]i triggered by 50 mM KCl could not be modulated by a sodium–calcium exchanger antagonist 5 mM NiCl2 alone, the increase of [Ca2+]i induced by BmK I could be completely eliminated by NiCl2 (Fig. 3C, n = 60–100, p < 0.05; Fig 3D, n = 60–100, p > 0.05). Moreover, it is noticed that BmK I was unable to modulate spontaneous Ca2+ release from SR induced by 10 mM caffeine in the absence of external Ca2+ (Fig. 4).
3. Discussion Our previous studies found that rat cardiac contractility could be increased significantly by BmK I, suggesting CICR from SR might be modulated by BmK I via either direct or indirect pathway. In cardiac muscle, CICR and contraction were usually triggered by Ca2+ influx via pathway of L-type
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Ca2+ channels. Additionally, T-type Ca2+ channels and the reverse mode Na+–Ca2+ exchanger could also contribute to extracellular Ca2+ entry across the plasmalemma. However, T-type Ca2+ channels are negligible in most rat ventricular myocytes (Nuss and Houser, 1993). In this study, it was found that BmK I could not work on L-type Ca2+ channels, thus ruling out the possibility that Ca2+ influx increase via Ca2+ channels directly triggers SR Ca2+ release. It has been demonstrated that tetrodotoxin/saxitoxin associating with receptor-site 1 on VGSCs could block Na+ channels, thus resulted in the decrease of contractile force, whereas a-scorpion and sea anemone toxins specifically binding to receptor-site 3 on VGSCs could modulate the inactivation phase of Na+ channels, and then led to increase in contractility. Therefore, VGSCs are critical to the negative or positive inotropism induced by above neurotoxins (Barnes and Hill, 1988; Wang and Strichartz, 1982). In this study, it was firstly found that the inactivation phase of Na+ currents was strongly delayed while peak Na+ currents did not be modulated by BmK I. This result is in agreement with the notion that scorpion a-toxin typically only slightly increases or decreases the peak sodium currents (Gordon et al., 1996). Since the voltage dependence of activation and inactivation was not altered strikingly, and time constants of inactivation were about 10-fold larger than normal, one can conclude that BmK I results in an uncoupling of activation and inactivation by binding to receptor site 3. It was well known that few sodium channels open at resting potential. The depolarization of cell membrane by 50 mM KCl results in a shift of membrane potential close to 20 mV accompanied by the activation of a substantial sodium channels. Consequently, slowing of sodium channel inactivation by BmK I may evoke more Na+ influx and produce a huge effect (see Fig. 2B). Unexpectedly, the fluorescence digital imaging found that 10 lM of the BmK I was less effective than that at the dose of 1 lM (see Fig. 2B). We postulate that there might be some complex mechanisms underlying the modulation of BmK I on the intracellular Na+ through the inactivation process of Na+ channels in cardiac myocytes. In addition, the change in internal Na+ depends on the number of channels open and the driving force for Na+, which could responsible for this phenomenon. A contribution of Na+ channels to E–C coupling usually dose not mean that Na+ itself triggers Ca2+ release from the SR but requires the reverse mode Na+–Ca2+ exchanger as an intermediate step after Na+ accumulation. The ability of Na+–Ca2+ exchanger to trigger Ca2+ release from SR has been defined in cardiac ventricular myocytes of rat, guinea pig and rabbit (Bers et al., 1988; Nuss and House, 1992; Carmeliet, 1992; Levi et al., 1993; Beuckelmann and Wier, 1988; Han et al., 2002; Wasserstrom and Vites, 1996; Sipido
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Fig. 3. The increase of the [Ca2+]i modulated by BmK I in a dose-dependent manner. (A) The pseudo color graph of F340/F380 ratio in Fura-2/AM loaded cardiac myocytes in the presence of either 50 mM KCl or 50 mM KCl + 500 nM BmK I. Top graph represented peak ratio change after administration of 50 mM KCl, the bottom figure showed that pseudo color varied from yellow to red induced by 50 mM KCl + 500 nM BmK I, indicating that the value of ratio shifts from low level to high one. Accordingly, the normalized ratio was enhanced in a dose-response manner as shown in (B) *p < 0.01, **p < 0.001 by StudentÕs t-test. (C) The [Ca2+]i was evoked by BmK I in the presence of 5 mM NiCl2. *p < 0.05 vs KCl. # p < 0.05 vs NiCl2. The increase of [Ca2+]i was completely abolished by 5 mM NiCl2. (D) Free [Ca2+]i triggered by 50 mM KCl could not be modulated in the presence of 5 mM NiCl2 alone. Error bars represent mean ± SD (n = 60–100).
Fig. 4. Spontaneous Ca2+ transients from SR evoked by 10 mM caffeine in the presence of BmK I. (A) SR Ca2+ release (F340/F380) in Fura-2 loaded cells was induced by 10 mM caffeine in the absence of external Ca2+ and presence of 3 mM EGTA. (B) SR Ca2+ release induced by 10 mM caffeine could not be modulated by 500 nM BmK I in the absence of external Ca2+.
et al., 1997; Litwin et al., 1998). In this study, free [Ca2+]i triggered by 50 mM KCl could not be modulated by a sodium–calcium exchanger antagonist 5 mM NiCl2 alone, but the significant increase of [Ca2+]i induced by BmK I could be completely abolished by NiCl2. This suggested that the Na+–Ca2+ exchanger did play an important role in the increase of [Ca2+]i, which might be responsible for the rat heart contractility increase induced by BmK I. In addition, it could be excluded that the ability of NiCl2 to abolish INa induced [Ca2+]i transients might be attributed to the blockade of T-type
Ca2+ channels for T-type Ca2+ channels are negligible in most rat ventricular myocytes. It was also noticed that the normalized ratio of [Na+]i was increased by only 8% even with 1 lM BmK I, but [Ca2+]i was greatly expanded by 23% with the same applied dose. It strongly indicated that the gain of Ca2+ release might be controlled by [Na+]i in subspace. Besides, the requirement for [Ca2+]o rules out the possibility that SR Ca2+ release induced by BmK I is tightly coupled to Na+ current activation. Thus, release of SR Ca2+ induced by BmK I most likely involves a CICR mechanism. On the other hand, BmK I has been demonstrated to be capable of blocking VGSCs inactivation process in crayfish giant axon (Terakawa et al., 1989), and binding to a single class of noninteracting receptor site on insecttype VGSCs, but lacking specific binding site on rat brain-type VGSCs (Li and Ji, 2000). The specific-modulating of BmK I on VGSCs of rat cardiac myocytes examined in the study suggests that the receptor site 3 on VGSCs of cardiac myocytes, responsible for the channel inactivation and specific associating with ascorpion toxins, is similar to site 3 on insect/mammal peripheral VGSCs, but distincts from that on rat brain type VGSCs. The specific-binding of BmK I to receptor site 3 of VGSCs suggested that BmK I would be valuable tool for identifying and characterizing the properties of VGSCs subtypes in addition to TTX.
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Finally, we conclude that: (a) cardiac VGSCs are also the targets of BmK I, and participate rat heart E–C coupling; (b) the reverse-mode Na+–Ca2+ exchanger triggered by Na+ accumulation plays a major role in augment of rat heart contractility induced by BmK I; (c) the receptor site 3-specific associating with BmK I on either cardiac VGSCs or insect/mammal peripheral VGSCs may share a functional architecture feature. Acknowledgements This work was supported by National Basic Research Program of China (1999054001), partially grants from National Nature Sciences Foundation of China (39625010). The authors thank Dr. Hai-Feng Zhu for skillful technical assistance, and Hamamatsu Photonics K.K., Japan for providing cool-charge coupled system. This article is also presented in memory of Professor Noboru Yanaihara. References Almeida, A.P., Alpoim, N.C., Freire-Maia, L., 1982. Effects of a purified scorpion toxin (Tityustoxin) on the isolated guinea pig heart. Toxicon 20 (5), 855–865. Barnes, S., Hill, B., 1988. Veratridine modifies open sodium channels. Journal of General Physiology (London) 91, 421–443. Benn, S.C., Costigan, M., Tate, S., Fitzgerald, M., Woolf, C.J., 2001. Developmental expression of the TTX-resistant voltage-gated sodium channels Nav1.8 (SNS) and Nav1.9 (SNS2) in primary sensory neurons. Journal of Neuroscience 21 (16), 6077–6085. Bers, D.M., Christensen, D.M., Nguyen, T.X., 1988. Can Ca entry via Na–Ca exchange directly activate cardiac muscle contraction? Journal of Molecular Cellular Cardiology 20, 405–414. Beuckelmann, D.J., Wier, W.G., 1988. Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells. Journal of Physiology 405, 233–255. Carmeliet, E., 1992. A fuzzy subsarcolemmal space for intracellular Na+ in cardiac cells? Cardiovascular Research 26, 433–442. Catterall, W.A., 2000. From ionic currents to molecular mechanisms: The structure and function of voltage-gated sodium channels. Neuron 26, 13–25. Cestele, S., Catterall, W.A., 2000. Molecular mechanisms of neurotoxin action on voltage-gated sodium channels. Biochimie 82, 883– 892. Cestele, S., Stankiewicz, M., Mansuelle, P., De, W.M., Dargent, B., Gilles, N., Pelhate, M., Rochat, H., Martin, E.M.F., Gordon, D., 1999. Scorpion alpha-like toxins, toxic to both mammals and insects, differently interact with receptor site 3 on voltage-gated sodium channels in mammals and insects. European Journal of Neuroscience 11, 975–985. Chen, B., Wang, C.Y., Ji, Y.H., 2001. Scorpion BmK venom induces nociceptive response of rat by plantar injection. Neurotoxicology and Teratology 23, 675–679. Decosterd, I., Ji, R.R., Abdi, S., Tate, S., Woolf, C.J., 2002. The pattern of expression of the voltage-gated sodium channels Nav1.8 and Nav1.9 dose not change in uninjured primary sensory neurons in experimented neuropathic pain models. Pain 96, 269–277. Denac, H., Mevissen, M., Scholtysik, G., 2000. Structure, function and pharmacology of voltage-gated sodium channels. Naunyn Schmiedebergs Arch Pharmacology 362, 453–479.
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Satoh, H., Hayashi, H., Noda, N., Terada, H., Kobayashi, A., Hirano, M., Yamashita, Y., Yamazaki, N., 1994. Regulation of [Na+]i and [Ca2+]i in guinea pig myocytes: Dual loading of fluorescent indicator SBFI and fluo 3. American Journal of Physiology (Heart Circ Physiol 35) 266, H568–H576. Sham, J.S.K., Cleemann, L., Morad, M., 1992. Gating of the cardiac Ca2+ release channel––the role of Na+ current and Na+–Ca2+ exchange. Science 255, 850–853. Sipido, K.R., Maes, M., Werf, F.V., 1997. Low efficiency of Ca2+ entry through Na+–Ca2+ exchange as trigger for Ca2+ release from the sarcoplasmic reticulum. Circulation Research 81, 1034–1044. Song, L.-S., Sham, J.S., Stern, M.D., Lakatta, E.G., Cheng, H., 1998. Direct measurement of SR release flux by tracking ÔCa2+Õ in rat cardiac myocytes. Journal of Physiology 512, 677–691. Sun, H.Y., Zhu, H.F., Ji, Y.H., 2003. BmK I, an alpha-like scorpion neurotoxin, specifically modulates isolated rat cardiac mechanical and electrical activity. Acta Physiologica Sinica 55 (5), 530–534. Tan, Z.Y., Xiao, H., Mao, X., Wang, C.Y., Zhao, Z.Q., Ji, Y.H., 2001. The inhibitory effects of BmK IT2, a scorpion neurotoxin on rat
nociceptive flexion reflex and possible mechanism of modulating voltage-gated Na+ channels. Neuropharmacology 40 (3), 352–357. Terakawa, S., Kimura, Y., Hsu, K., Ji, Y.H., 1989. Lack of effect of a neurotoxin from the scorpion Buthus martensi Karsch on nerve fibers of this scorpion. Toxicon 27 (5), 569–578. Tyrrell, L., Renganathan, M., Sulayman, D., Dib, H., Waxman, S.G., 2001. Glycosylation alters steady-state inactivation of sodium channel Nav1.9 in dorsal root ganglion neurons and is developmentally regulated. Journal of Neuroscience 21 (24), 9629– 9637. Vargas, O., Martin, M.F., Rochat, H., 1987. Characterization of six toxins from the venom of the Moroccan scorpion Buthus occitanus mardochei. European Journal of Biochemistry 162, 589–599. Wang, G.K., Strichartz, G., 1982. Simultaneous modifications of sodium channel gating by two scorpion toxins. Biophysical Journal 40, 175–179. Wasserstrom, J.A., Vites, A.M., 1996. The role of Na+–Ca2+ exchange in the activation of excitation–contraction coupling in rat ventricular myocytes. Journal of Physiology 493, 529–542.
The role of voltage-gated Na+ channels in excitation–contraction coupling of rat heart determined by BmK I, an a-like scorpion neurotoxin Hai-Ying Sun a
a,b
, Zhao-Nian Zhou a, Yong-Hua Ji
a,c,*
The Key Laboratory of Neurobiology, Institute of Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, PR China b School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200030, PR China c School of Life Sciences, Shanghai University, Shanghai 200436, PR China Received 31 December 2003; accepted 7 July 2004
Abstract A mechanism underlying the increase in rat heart contractility modulated by BmK I, an a-like scorpion neurotoxin, was investigated using whole-cell patch-clamp and fluorescence digital imaging techniques. Results showed that (a) L-type Ca2+ current could not be modified by 500 nM BmK I; (b) The inactivation process of Na+ current was significantly delayed with no change of its amplitude; (c) The overall intracellular Na+ and Ca2+ concentration could be augmented in the presence of BmK I; (p < 0.05); (d) The increase of free intracellular Ca2+ concentration induced by BmK I was inhibited completely by 5 mM NiCl2 (p < 0.05), an inhibitor of Na+–Ca2+ exchanger; (e) The spontaneous Ca2+ release induced by 10 mM caffeine from sarcoplasmic reticulum could not be modulated by 500 nM BmK I in the absence of external Ca2+. These results indicate that cardiac voltage-gated Na+ channels are also targets of BmK I. Na+ accumulation through Na+ channels can trigger sarcoplasmic reticulum Ca2+ release in rat cardiac myocytes via reverse-mode Na+–Ca2+ exchanger. Furthermore, Ca2+ release from sarcoplasmic reticulum induced by BmK I most likely involves a Ca2+-induced release mechanism. 2004 Elsevier Ltd. All rights reserved. Keywords: Inotropism; Voltage-gated Na+ channel; Reverse-mode Na+–Ca2+ exchanger; BmK I
Voltage-gated Na+ channels (VGSCs) composed of pore-forming a and b subunits are responsible for the generation and propagation of action potential in excitable cells (Catterall, 2000). Nine genes encoding a subunits have been functionally expressed. These isoforms can be differentiated by their distinct patterns of development and localization in the nervous system, skeletal and cardiac muscle, as well as the relative sensitivity to tetrodotoxin (TTX) (Gellens et al., 1992; Goldin et al., 2000). Isoforms expressed in the central nervous system *
Corresponding author. Tel.: +86 21 54920300; fax: +86 21 54920276. E-mail address: [email protected] (Y.-H. Ji). 0887-2333/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2004.07.005
(Nav1.1, 1.2, 1.3 and 1.6) are inhibited by nanomolar concentrations of TTX, as is the isoform present in adult skeletal muscle (Nav1.4). In contrast, the primary cardiac isoform (Nav1.5) requires micromolar concentrations of TTX for inhibition (Goldin, 2001). Recently, VGSCsmediated cardiac calcium-induced calcium release (CICR) and contraction have been extensively investigated in cat and guinea pig ventricular cells. However, the results obtained from different experimental protocols seem to be controversial (Levi et al., 1993; Renaud et al., 1986; Sham et al., 1992; Lipp and Niggli, 1994). Thus, the study on how VGSCs (Nav1.5) modulate heart contractility is worthwhile to pursue to understand excitation–contraction (E–C) coupling in rat heart.
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It is well known that VGSCs are the targets of numerous natural neurotoxins, which have been extensively examined to approach to the gating mechanism of VGSCs involved in neuronal signaling transmission such as algesia and analgesia (Josephine et al., 2002; Decosterd et al., 2002; Tyrrell et al., 2001; Benn et al., 2001; Gold, 1999; Cestele and Catterall, 2000; Denac et al., 2000; Tan et al., 2001). Most long-chain neurotoxic polypeptides purified from the scorpion venoms are also determined to be VGSCs-specific modulators. Generally, they are classified into two main classes, the a- and b-toxins according to their modulating effects on Na+ currents and binding characteristics to VGSCs (Gordon et al., 1998). Among them, BmK I was one of the most toxic component purified from the venom of Chinese scorpion Buthus martensi Karsch (BmK) with nociceptive-inducing activity (Ji et al., 1996; Chen et al., 2001). BmK I could favorably modulate the inactivation phase of VGSCs in crayfish axons in the same mode as classic a-toxins (Terakawa et al., 1989), and also could interact with insect VGSCs that distinguished it from classical a-toxins such as AaH II (Gordon et al., 1996). It is surprising that BmK I was unable to compete with the binding of either a- or b-toxins to receptor site 3 and 4 of mammal brain-type Na+ channels. Therefore, it is considered to belong to the a-like group (Cestele et al., 1999; Vargas et al., 1987; Li and Ji, 2000). Although the modulating activity of a few neurotoxins on either guinea pig or rat heart contractile force and the Na+ currents have been demonstrated, such as TsTX and TiTXc from Brazilian scorpion Tityus serrulatus, and AxII from Anthopleura xanthogrammica (Almeida et al., 1982; Marcotte et al., 1997; Renaud et al., 1986; Goudet et al., 2001), it remains to be unraveled whether a-like scorpion neurotoxin BmK I is sensitive to rat cardiac VGSCs, and how VGSCs are involved in signaling pathway of rat cardiac excitation–contraction (E–C) coupling so far. Our previous study indicated that BmK I could increase rat heart contractility in a dose-dependent manner (Sun et al., 2003). The present study was designed to investigate the modulating effects of BmK I on VGSCs in rat cardiac myocytes, and to determine the potential mechanisms underlying the increase of rat cardiac contractility. Furthermore, it might provide insight into the receptor sites-specific association with BmK I between neuronal and cardiac VGSCs.
moved and perfused retrogradely through the aorta with Ca2+-free TyrodeÕs solution at 37 C, followed by the Ca2+-free TyrodeÕs solution containing 50 lM Ca2+, 1% BSA and 0.04% type II collagenase (Sigma, USA) for 5–10 min. After perfusion, the separated ventricles were chopped into small pieces, incubated in fresh TyrodeÕs solution for 10 min. The isolated cells were filtrated through a nylon mesh (200 lm) and suspended in the TyrodeÕs solution, in which the Ca2+ concentration gradually increased to 1.0 mM. Only the cells showed rod shaped and clear cross striation were used for the experiments. The Ca2+-free TyrodeÕs solution contained: NaCl (135 mM), KCl (5.4 mM), MgCl2 (1.0 mM), NaH2PO4 (0.33 mM), HEPES (5.0 mM), and Glucose (10 mM) adjusted to pH 7.4 with 0.5 N NaOH. 1.2. Recording of INa and ICa The whole-cell patch clamp technique was employed to record INa and ICa in single ventricular myocyte. All recordings were carried out at room temperature (20– 22 C). The cells were constantly perfused after adhering to the bottom of the chamber. Membrane currents were recorded with EPC9 amplifier (HEKA elektronik, Germany) controlled by EPC9 data acquisition system. The microelectrodes fabricated with microelectrodes puller (PP-830, Narishige, Japan) had a resistance of 2–3 MX when filled with pipette solution. In the whole cell configuration, the series resistance was partially compensated by about 70–80%. Currents were filtered at 5 kHz using a four-pole low-pass Bessel filter. Digital leak subtraction of the current records was carried out by a P/4 protocol. The bath solution of recording INa was composed of (mM): NaCl 90, Choline-Cl 40, CsCl 5.4, HEPES 10, MgCl2 1, NaH2PO4 0.33, CaCl2 1.8, Glucose 10 (pH adjusted with CsOH to 7.4). The pipette solution was composed of (mM): CsCl 20, Cs-fluoride 110, NaCl 5, HEPES 5, EGTA 5, MgCl2 1, Na2-ATP 5 (pH adjusted to 7.2 with CsOH). The bath solution of recording ICa was composed of (mM): Choline-Cl 135, CsCl 5.4, HEPES 10, MgCl2 1.0, NaH2PO4 0.33, CaCl2 1.8, Glucose 10 (pH adjusted with CsOH to 7.4). The pipette solution of recording ICa was composed of (mM): CsCl 20, Cs-aspartate 110, HEPES 5, EGTA 10, MgCl2 1.0, Mg2-ATP 5 (pH adjusted to 7.2 with CsOH). 1.3. Loading of Myocytes with Fura-2/AM and measurement of free [Ca2+]i
1. Materials and methods 1.1. Preparation of adult rat ventricular myocytes Cardiac ventricular myocytes were isolated from Sprague–Dawley adult rats using a standard enzymatic technique (Song et al., 1998). Briefly, rat hearts were re-
Single ventricular myocytes were loaded with 2.5 lM fura-2 acetoxymethyl ester mixed to 0.1% Pluronic F127 (Sigma Chemical Co., USA) in TyrodeÕs solution containing 1.8 mM CaCl2 for 30 min at 37 C. The loaded myocytes were then washed twice with TyrodeÕs solution, and incubated for at least 30 min at room temper-
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ature to completely hydrolyze the dye. The fluorescence intensity of dye-loaded cells was 2–3 times higher than background fluorescence with unloaded cells. The background noise could be automatically corrected and subtracted at each experiment in cool-charge coupled system (C4742-95-12NRB, Hamamatsu Photonics K.K., Japan). The coverslip with the loaded cells was introduced into a transparent perfusion chamber positioned in the light path of an inverted microscope (IX-70, Olympus Optical CO., Tokyo, Japan). Alternating excitation of the fluorescent dye at wavelengths of 340 and 380 nm for fura-2 was performed with an AR-Cation measurement system adapted to the microscope. Emitted light (510 ± 7.5 nm) was detected by cool-charge coupled system. The light signal was recorded and analyzed by software of Aquacosmos 1.2. Additions of agents were made through a MASTERFLEX pumps to ensure optimum performance. Because of the inherent problems with calibration of the fura-2 ratio, data were generally expressed in arbitrary units of fluorescence ratio. The ratio = (F340 F340base)/(F380 F380base). For each experiment, 5–7 groups (cells in different coverslips) data (total cell number ranges from 60 to 100) were summed in every group first, and then each sum was averaged by the cell number. The averaged ratio of each group was normalized, and then the normalized ratios of different groups were summed and averaged by the group number. Moreover, the pseudo color figures of fluorescence ratio before and after administration of BmK I were collected. All experiments were performed at 35 C. 1.4. Loading of myocytes with SBFI/AM and measurement of [Na+]i Single ventricular myocytes were loaded with 10 lM benzofuran isophthalate (SBFI-AM) in the form of its acetoxymethyl ester mixed to 0.1% Pluronic F127 (Sigma Chemical Co., USA) in TyrodeÕs solution containing 1.8 mM CaCl2 for 30 min at 37 C. The rest manipulations were the same as that of the free [Ca2+]i. 1.5. The calibration of SBFI fluorescence The calibration of SBFI fluorescence is performed according to the method described previously (Satoh et al., 1994). Briefly, the cells were loaded with SBFI and then perfused with calibration solutions with various Na+ concentration (0, 5, 10, 30, 50 mM). The calibration solutions were made from appropriate mixtures of high Na+ and high K+ solutions. The high Na+ solution contained (in mM): sodium gluconate 110, NaCl 30, MgCl2 0.6, Na-HEPES 10, EGTA 1, pH was adjusted to 7.1 by adding HCl. The composition of high K+ solution was identical to that of high Na+ solution except complete replacement of Na+ by K+.
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Ten micromoles gramicidin D and 100 lM strophanthidin were added. The cells were incubated with each solution for at least 10 min to allow complete equilibration. [Na+]i = Kd · b · (R Rmin)/(Rmax R). R is the obtained 340/380 nm excitation ratio, Rmin is the ratio at 0 mM [Na+]i, Rmax is the ratio at saturating [Na+]i, Kd is the dissociation constant, and b is the ratio of the excitation efficient of free to Na+ bound SBFI at 380 nm. 1.6. Scorpion toxin The purified toxin BmK I was provided by our laboratory (Ji et al., 1996). 1.7. Statistics All averaged and normalized data were presented as mean ± SD. The statistical significance of the differences in the calculated mean values was evaluated using the StudentÕs t-test. P-value is provided in the text. Statistical significance was assumed when P < 0.05.
2. Results 2.1. Delay of inactivation process of INa modulated by BmK I Sodium currents were evoked by step depolarization of 25 ms ranging from 70 to 0 mV, with increments of 10 mV, from a holding potential of 80 mV. Na+ currents inactivated rapidly after activation in control condition (Fig. 1A left panel, n = 6). In the presence of 500 nM BmK I, the inactivation kinetics were potently slowed down at all tested pulse without the change of peak Na+ currents (Fig. 1A right panel, n = 6), just like scorpion a-toxins (Lee et al., 2000). Current–voltage (I– V) relation was determined using 25-ms depolarizing steps every 10 s from 70 mV to +30 mV with a holding potential (HP) of 80 mV. In order to allow for differences in myocytes size, the magnitude of peak INa currents was normalized to cell capacitance (pA/pF). Cell capacitance of myocytes used in our experiments was 71.33 ± 18.61 pF, the series resistance (Rs) was 5.32 ± 1.06 MX (n = 10) during recording process. Results showed that the density of INa in rat ventricular myocytes was increased slightly from 28.77 ± 3.12 pA/ pF to 30.19 ± 3.89 pA/pF after exposure to 500 nM BmK I (Fig 1B n = 10 from six rats, p > 0.05). On the other hand, calcium current was evoked by a step depolarization to 0 mV during 300 ms from a holding potential of 40 mV. Fig 1C displayed that L-type calcium current at 0 mV could not be modified after the application of 500 nM BmK I (n = 6, p > 0.05). Steady-state activation of INa was evaluated by fitting normalized conductance–voltage values to the Boltzman
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Fig. 1. Electrophysiological recording of rat ventricular myocytes Na+ currents and Ca2+ current modulated by BmK I. (A) Representative tracing of Na+ currents evoked by depolarization ranging from 70 mV to 0 mV by 10 mV steps from a holding potential of 80 mV in the absence (left panel) or presence (right panel) of 500 nM BmK I (n = 6). Inactivation phase of Na+ currents was prolonged significantly by 500 nM BmK I. (B) I–V curve of Na+ currents remained the same in the absence or presence of 500 nM BmK I (n = 6). (C) Representative L-type calcium current evoked by a step depolarization to 0 mV during 300 ms from a holding potential of 40 mV in the absence or presence of 500 nM BmK I indicated by arrow. Ltype calcium current could not be modulated by 500 nM BmK I (n = 6). (D) Averaged and normalized steady-state activation curve of Na+ channel in the absence (j) or presence (d) of 500 nM BmK I. Steady-state activation of Na+ channel could not be modulated by 500 nM BmK I (n = 6). (E) Averaged and normalized curve of steady-state inactivation of Na+ channel was shifted slightly toward negative potential in the presence of 500 nM BmK I (n = 6, p > 0.05). Data stand for mean ± SD.
equation, g/gmax = {1/[1 + exp] (V Vh)/s]} 1. V is the test potential, Vh the midactivation or midinactivation potential, and s the slope factor. The activation curve of INa after addition of 500 nM BmK I was nearly overlapping with that of the control (Fig. 1D, n = 6). In the control, the voltage for half-maximal activation (V1/2) and the slope factor of the activation curve were 40.5 ± 1.0 mV and 5.44 ± 0.6 mV, respectively. In the presence of 500 nM BmK I, V1/2 and slope factor were 41.4 ± 1.1 mV and 4.94 ± 0.6 mV, respectively. Steady-state inactivation of INa was determined with a double-pulse protocol consisting of 300 ms conditioning pulse ranging from 120 to 30 mV, and followed by a constant test pulse of 50 ms duration to 30 mV at a pulsing frequency 0.5 Hz. The amplitude of peak INa during test pulse was normalized to the maximum peak current and plotted as function of the conditioning potential. Data was fitted to the Boltzman equation. In control, Vh and slope factor were 55.03 ± 2.1 mV and
15.09 ± 2.0 mV, respectively. In the presence of 500 nM BmK I, the Vh and slope factor were 58.87 ± 2.3 mV and 16.76 ± 2.3 mV (Fig. 1E, n = 6). Five hundred nanomoles BmK I shifted the I–V relation more hyperpolarized to 3.8 mV at which half of the cardiac Na+ channels were inactivated (n = 6, p > 0.05). In comparison with the time constant of inactivation under control condition, the time course of the inactivation phase s at 30 mV was increased about 10-fold in the presence of 500 nM BmK I from 1.53 ± 0.76 ms to 14.43 ± 2.45 ms (n = 6, p < 0.05). 2.2. Increase in [Na+]i and [Ca2+]i evoked by BmK I Treatment with 1 lM BmK I could cause a gradual rise in [Na+]i from normalized ratio of 1.00–1.08 during a 30-min duration, whereas the normalized ratio did not change under control conditions. The cells incubated with normal external solution only displayed a slight in-
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Fig. 2. Augment of the intracellular free Na+ evoked by BmK I. BmK I was applied at the time indicated by arrow. SBFI fluorescence ratio was proportional to the [Na+]i. (A) Calibrated [Na+]i concentration curve. (B) Intracellular Na+ was increased by BmK I. The number of cells varied from 60 to 100 in each group. *p < 0.05 vs control group 20 min after administration of 1 lM BmK I.
crease of [Na+]i from 1 to 1.005. One micromole BmK I could evoke maximal increase of [Na+]i (Fig. 2B, n = 60– 100, p < 0.05). In order to observe the changes of [Ca2+]i, 50 mM KCl was applied to make a depolarization and trigger CICR. Compared to control, [Ca2+]i was significantly elevated by BmK I in a dose-dependent manner (Fig. 3B, n = 60–100, p < 0.05). Although free [Ca2+]i triggered by 50 mM KCl could not be modulated by a sodium–calcium exchanger antagonist 5 mM NiCl2 alone, the increase of [Ca2+]i induced by BmK I could be completely eliminated by NiCl2 (Fig. 3C, n = 60–100, p < 0.05; Fig 3D, n = 60–100, p > 0.05). Moreover, it is noticed that BmK I was unable to modulate spontaneous Ca2+ release from SR induced by 10 mM caffeine in the absence of external Ca2+ (Fig. 4).
3. Discussion Our previous studies found that rat cardiac contractility could be increased significantly by BmK I, suggesting CICR from SR might be modulated by BmK I via either direct or indirect pathway. In cardiac muscle, CICR and contraction were usually triggered by Ca2+ influx via pathway of L-type
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Ca2+ channels. Additionally, T-type Ca2+ channels and the reverse mode Na+–Ca2+ exchanger could also contribute to extracellular Ca2+ entry across the plasmalemma. However, T-type Ca2+ channels are negligible in most rat ventricular myocytes (Nuss and Houser, 1993). In this study, it was found that BmK I could not work on L-type Ca2+ channels, thus ruling out the possibility that Ca2+ influx increase via Ca2+ channels directly triggers SR Ca2+ release. It has been demonstrated that tetrodotoxin/saxitoxin associating with receptor-site 1 on VGSCs could block Na+ channels, thus resulted in the decrease of contractile force, whereas a-scorpion and sea anemone toxins specifically binding to receptor-site 3 on VGSCs could modulate the inactivation phase of Na+ channels, and then led to increase in contractility. Therefore, VGSCs are critical to the negative or positive inotropism induced by above neurotoxins (Barnes and Hill, 1988; Wang and Strichartz, 1982). In this study, it was firstly found that the inactivation phase of Na+ currents was strongly delayed while peak Na+ currents did not be modulated by BmK I. This result is in agreement with the notion that scorpion a-toxin typically only slightly increases or decreases the peak sodium currents (Gordon et al., 1996). Since the voltage dependence of activation and inactivation was not altered strikingly, and time constants of inactivation were about 10-fold larger than normal, one can conclude that BmK I results in an uncoupling of activation and inactivation by binding to receptor site 3. It was well known that few sodium channels open at resting potential. The depolarization of cell membrane by 50 mM KCl results in a shift of membrane potential close to 20 mV accompanied by the activation of a substantial sodium channels. Consequently, slowing of sodium channel inactivation by BmK I may evoke more Na+ influx and produce a huge effect (see Fig. 2B). Unexpectedly, the fluorescence digital imaging found that 10 lM of the BmK I was less effective than that at the dose of 1 lM (see Fig. 2B). We postulate that there might be some complex mechanisms underlying the modulation of BmK I on the intracellular Na+ through the inactivation process of Na+ channels in cardiac myocytes. In addition, the change in internal Na+ depends on the number of channels open and the driving force for Na+, which could responsible for this phenomenon. A contribution of Na+ channels to E–C coupling usually dose not mean that Na+ itself triggers Ca2+ release from the SR but requires the reverse mode Na+–Ca2+ exchanger as an intermediate step after Na+ accumulation. The ability of Na+–Ca2+ exchanger to trigger Ca2+ release from SR has been defined in cardiac ventricular myocytes of rat, guinea pig and rabbit (Bers et al., 1988; Nuss and House, 1992; Carmeliet, 1992; Levi et al., 1993; Beuckelmann and Wier, 1988; Han et al., 2002; Wasserstrom and Vites, 1996; Sipido
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Fig. 3. The increase of the [Ca2+]i modulated by BmK I in a dose-dependent manner. (A) The pseudo color graph of F340/F380 ratio in Fura-2/AM loaded cardiac myocytes in the presence of either 50 mM KCl or 50 mM KCl + 500 nM BmK I. Top graph represented peak ratio change after administration of 50 mM KCl, the bottom figure showed that pseudo color varied from yellow to red induced by 50 mM KCl + 500 nM BmK I, indicating that the value of ratio shifts from low level to high one. Accordingly, the normalized ratio was enhanced in a dose-response manner as shown in (B) *p < 0.01, **p < 0.001 by StudentÕs t-test. (C) The [Ca2+]i was evoked by BmK I in the presence of 5 mM NiCl2. *p < 0.05 vs KCl. # p < 0.05 vs NiCl2. The increase of [Ca2+]i was completely abolished by 5 mM NiCl2. (D) Free [Ca2+]i triggered by 50 mM KCl could not be modulated in the presence of 5 mM NiCl2 alone. Error bars represent mean ± SD (n = 60–100).
Fig. 4. Spontaneous Ca2+ transients from SR evoked by 10 mM caffeine in the presence of BmK I. (A) SR Ca2+ release (F340/F380) in Fura-2 loaded cells was induced by 10 mM caffeine in the absence of external Ca2+ and presence of 3 mM EGTA. (B) SR Ca2+ release induced by 10 mM caffeine could not be modulated by 500 nM BmK I in the absence of external Ca2+.
et al., 1997; Litwin et al., 1998). In this study, free [Ca2+]i triggered by 50 mM KCl could not be modulated by a sodium–calcium exchanger antagonist 5 mM NiCl2 alone, but the significant increase of [Ca2+]i induced by BmK I could be completely abolished by NiCl2. This suggested that the Na+–Ca2+ exchanger did play an important role in the increase of [Ca2+]i, which might be responsible for the rat heart contractility increase induced by BmK I. In addition, it could be excluded that the ability of NiCl2 to abolish INa induced [Ca2+]i transients might be attributed to the blockade of T-type
Ca2+ channels for T-type Ca2+ channels are negligible in most rat ventricular myocytes. It was also noticed that the normalized ratio of [Na+]i was increased by only 8% even with 1 lM BmK I, but [Ca2+]i was greatly expanded by 23% with the same applied dose. It strongly indicated that the gain of Ca2+ release might be controlled by [Na+]i in subspace. Besides, the requirement for [Ca2+]o rules out the possibility that SR Ca2+ release induced by BmK I is tightly coupled to Na+ current activation. Thus, release of SR Ca2+ induced by BmK I most likely involves a CICR mechanism. On the other hand, BmK I has been demonstrated to be capable of blocking VGSCs inactivation process in crayfish giant axon (Terakawa et al., 1989), and binding to a single class of noninteracting receptor site on insecttype VGSCs, but lacking specific binding site on rat brain-type VGSCs (Li and Ji, 2000). The specific-modulating of BmK I on VGSCs of rat cardiac myocytes examined in the study suggests that the receptor site 3 on VGSCs of cardiac myocytes, responsible for the channel inactivation and specific associating with ascorpion toxins, is similar to site 3 on insect/mammal peripheral VGSCs, but distincts from that on rat brain type VGSCs. The specific-binding of BmK I to receptor site 3 of VGSCs suggested that BmK I would be valuable tool for identifying and characterizing the properties of VGSCs subtypes in addition to TTX.
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