© 1997 American Heart Association, Inc.
Articles |
Effects of Tityus serrulatus Scorpion Toxin 
on Voltage-Gated Na+ Channels
Laval Hospital (P.M., M.C.), Research Center, Quebec, Canada, and the Department of Biochemistry and Biophysics (L.-Q.C., R.G.K.), University of Pennsylvania School of Medicine, Philadelphia.
Correspondence to Dr M. Chahine, Laval Hospital, Research Center, 2725, Chemin Ste-Foy, Quebec, Canada, G1V 4G5. E-mail mohamed.chahine@phc.ulaval.ca
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Abstract |
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The effects of Brazilian scorpion Tityus serrulatus toxin
(TiTx) were studied on voltage-gated Na+ channels from human heart (hH1) and rat skeletal muscle (rSkM1). The Na+ channels were expressed in Xenopus laevis oocytes, and Na+ currents were recorded using two-microelectrode voltage-clamp techniques. In control experiments, the threshold of activation of hH1 is more negative than that of rSkM1 by 
20 mV. The toxin induces a shift of the voltage dependence of activation toward more negative potential values and reduces the amplitude of the current when administered to rSkM1. In contrast, TiTx has little discernible effect on the current-voltage curve for hH1 at 100 nmol/L. Chimeric channels formed from these two isoforms were constructed to localize the binding site of TiTx on rSkM1. TiTx shifts the activation of a chimera (SSHH) in which domains 1 (D1) and 2 (D2) derive from rSkM1 and domains 3 (D3) and 4 (D4) derive from hH1. This finding suggests that the toxin acts on the activation of rSkM1 by binding either to D1 and/or D2. TiTx shifted the activation of another chimera with D2-D3-D4 from rSkM1 (HSSS) toward more hyperpolarizing potentials and had no effect on the activation of other chimeras with only D1-D3-D4 from rSkM1 (SHSS) or only D3 from rSkM1 (HHSH). Finally, a chimera in which D2 is from rSkM1 and all others domains are from hH1 (HSHH) provides further compelling support for our hypothesis. TiTx shifts the activation of this chimera toward more negative potential values. Thus, TiTx action on chimeras segregates with the source of D2: when D2 is from rSkM1, the toxin affects activation. We infer that D2 plays an important role in the activation process of voltage-gated Na+ channels.
Key Words: sodium channel • Tityus serrulatus toxin • voltage clamp • rat skeletal muscle • human cardiac muscle
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Introduction |
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Voltage-gated NaChs are membrane proteins that play an essential role in establishing cell excitability and maintaining its conduction properties.1 2 3 In many excitable cells, NaChs are responsible for the rising phase of the action potential. There are many types of voltage-gated NaChs, each one with distinct kinetic, pharmacological, and immunological properties.3 hH14 5 and rSkM16 voltage-gated NaChs have been cloned, sequenced, expressed in heterologous systems such as Xenopus oocytes or mammalian cell lines, and characterized electrophysiologically and pharmacologically. Their primary sequence structures consist of four homologous domains (D1 to D4) linked by interdomains, intracellular loops; each domain contains six
-helical transmembrane segments (S1 to S6).7 This general structure is conserved among different cloned voltage-gated NaChs.8
A variety of natural toxins are known to act on voltage-gated NaChs by binding at specific high-affinity sites. These induce various effects and are widely used as powerful tools to characterize the molecular structure of NaChs.2 9 10 Toxins are classified into five groups based on their binding/receptor site characteristics on voltage-gated NaChs.2 Some toxins slow inactivation without affecting activation (eg, -scorpion toxin and sea anemone toxin ATX-II [class 3]), others function as channel blockers (eg, tetrodotoxin, saxitoxin, and µ-conotoxin [class 1]), and another group shifts the activation voltage dependence in the hyperpolarizing direction (eg, TiTx [class 4]). The premise is that locating the interaction sites will help determine which parts of the channel protein are involved in its various functions.
TiTx, a Brazilian scorpion toxin, which is the subject of this work, is a 64–amino acid peptide.11 Eleven of its amino acid residues are positively charged (Arg,Lys); five are negatively charged (Asp,Glu), giving the toxin a net charge of +6; and there are eight aromatic residues (Trp,Tyr). The gene encoding this toxin was cloned and contains an intron.11 TiTx is very potent, being effective at concentrations in the nanomolar range; this toxin has been reported to have the highest affinity for NaChs, with a Kd value of 2.3 pmol/L, obtained using binding studies on membrane synaptosomes.12 Experiments on rabbit and frog skeletal muscle showed that TiTx acts on NaChs in the sarcolemmal membrane but not on NaChs in the T-tubule membrane.13 This toxin reduces the amplitude of the peak INa, and it has been suggested that TiTx revealed a population of NaChs that activate at potentials 30 to 40 mV more negative than the NaChs in neuroblastoma cells.14 The shift of the voltage dependence of activation and the reduction of peak amplitude current in response to the toxin were also observed in cultured rat muscular cells15 and in a peripheral nerve membrane of Xenopus laevis.16 TiTx was shown to produce complex effects on the rate and the contractile force of isolated guinea pig heart, possibly due to the simultaneous release of acetylcholine and catecholamines from postganglionic nerve fibers in the heart.17 More recently, complex effects of TiTx were demonstrated on both the rate and the contractile force of the isolated rat atria.18 These effects might be related to an action of the toxin on the neuronal NaChs innervating the heart (eg, causing release of acetylcholine) rather than to an effect directly on the myocardial NaChs themselves. However, other authors have described effects of the same toxin on neonatal rat ventricular cells using single-channel and whole-cell patch-clamp experiments.19 The purpose of our experiments was to determine the effects of TiTx on hH1 and rSkM1 expressed in the heterologous Xenopus oocyte system. In this way, we avoid the ambiguities created by neural and endocrine influences. Since the sensitivity of hH1 and rSkM1 to the effect of TiTx on activation differs, we used chimeras formed from these two isoforms in order to localize the site of toxin action on rSkM1.
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Materials and Methods |
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Expression in Xenopus Oocytes
The preparation of Xenopus oocytes was described elsewhere.20 21 Briefly, stage V or VI oocytes were injected with hH1, rSkM1, or chimeric NaCh cRNA. The oocytes were maintained at 18°C in a diluted solution of Leibovitz's L-15 medium (GIBCO) enriched with 15 mmol/L HEPES (pH 7.6), 1 mmol/L glutamine, and 50 µg/µL gentamycin. Oocytes were used for experiments 1 to 3 days after the injection.
Electrical Recording
The macroscopic INas from the cRNA-injected oocytes were measured by two 3 mol/L KCl–filled microelectrodes in voltage-clamp experiments. Membrane potential was controlled by a Warner oocyte clamp (Warner Instrument Corp). A ground metal shield was inserted between the two microelectrodes to minimize electrode coupling and to speed the clamp rise time. The total volume of the bath was 400 µL. Bathing solution changes required <5 s. Voltage commands were generated by computer using pCLAMP software (version 5.5, Axon Instruments). INas were elicited from a holding potential of -100 mV in 5-mV increments from -80 to +25 mV. Threshold of the INas (appearance of inward currents) was detected using a cursor (Clampan, pCLAMP software, version 5.5, Axon Instruments). Below -70 mV, no inward INas were detected for both hH1 and rSkM1; however, at more positive membrane potentials, inward INas were detected, and their amplitudes were measured. Kinetics of inactivation were studied by measuring the time for the current to decay to 0.5 of its value at the peak (t½). Currents were filtered at 2 kHz (-3 dB, four-pole Bessel filter). Data are expressed as mean±SEM.
Solutions
The bathing solution was a Ringer's solution that contained (mmol/L) NaCl 116, KCl 2, CaCl2 1.8, MgCl2 2, and HEPES 5; pH was adjusted to 7.6 at 22°C with NaOH. TiTx was a gift from Dr Michel Lazdunski, Centre de Biochimie du Centre National de la Recherche Scientifique, Nice, France. TiTx was purified in Dr Michel Lazdunski's laboratory as described previously.12 Stock solutions were made in water at 10-3 mol/L and stored at -20°C.
Recombinant DNA Constructions of Chimeric Channels
pS1S1 (pSelect-1/rSkM1) has been described previously.22 The pS1S1' is a cassetted version of pS1S1 with Sal I, Sac I, Hpa I, and Afl II sites introduced as silent mutations at nucleotides 2171, 2843, 3967, and 4568, respectively, using the following antisense oligonucleotides: Sal I, 5'-TACCCAGGTCGACAAAGGGGT-3'; Sac I, 5'-CACTGAAGGAGCTCAGCAGGA-3'; Hpa I, 5'-CAAACAAGTTAACCCCCATGA-3'; and Afl II, 5'-TGTCCACCTTAAGCTGGCTCT-3'. The underlined nucleotides are the mutation sites.
pS1H1 (pSelect-1/hH1) has been described previously.22 pS1H1' is a cassetted version of pS1H1 with Spl I, Sal I, Sac I, Hpa I, and Afl II introduced as silent mutation at nucleotides 1394, 2305, 2967, 4209, and 4813, respectively, using the following antisense oligonucleotides: Spl I, 5'-TTTGCTCCTCGTACGCCATTGCGA-3'; Sal I, 5'-TGATGGTGAGGTCGACAAACGGGTCCATG-3'; Sac I, 5'-TGAAGGAGCTCAGCAGCAAGG-3'; Hpa I, 5'-CAAAGAGGTTAACGCCCATGA-3'; and Afl II, 5'-GGCCAAGATGTTGATCTTAAGAGGACTTTGGTCATC-3'.
pcDNA/HSSS (11.1 kb) consists of pcDNA1 (5.4 kb with Hind III and Not I ends), Hind III–Spl I (1.2 kb from pS1H1'), and Spl I–Not I (4.5 kb from pS1S1') fragments. The junction sequence is as follows:
pcDNA/HHSH (11.5 kb) consists of pcDNA1 (5.4 kb with Hind III and EcoRI ends), Hind III–Sac I (2.8 kb from pS1H1'), Sac I–Hpa I (1.1 kb from pS1S1'), and Hpa I–EcoRI (3.3 kb from pS1H1') fragments. The junction sequences are as follows:
pcDNA/SSHH (12.4 kb) consists of pcDNA1 (5.4 kb with Hind III and EcoRI ends), Hind III–Sac I (2.5 kb from pS1S1'), and Sac I–EcoRI (4.5 kb from pS1H1') fragments. The junction sequence is as follows:
pSelect-1/HSHH (11.3 kb) consists of Sal I–Sac I (0.6 kb from pS1H1') and Sal I–Sac I (10.7 kb from pS1S1') fragments. The junction sequences are as follows:
pcDNA/SHSS (11.1 kb) consists of pcDNA1 (5.4 kb with Hind III and EcoRI ends), Hind III–Sal I (1.8 kb from pS1S1'), Sal I–Sac I (0.6 kb from pS1H1'), Sac I–Hpa I (1.1 kb from pS1S1'), Hpa I–Afl II (0.6 kb from pS1H1'), and Afl II–EcoRI (1.6 kb from pS1S1') fragments. The junction sequences are as follows:
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Voltage-Gated NaCh Isoforms rSkM1 and hH1 Have Different Voltage Thresholds of Activation
Xenopus oocytes were injected with cRNA encoding human heart (hH1) and rat skeletal muscle (rSkM1) NaChs. INas were recorded 1 to 3 days later from a holding potential of -100 mV in 5-mV increments from -80 to +25 mV. Inward INas appeared at -40.6±0.9 mV (n=10) for rSkM1 and at -55.5±0.8 mV (n=11) for hH1 (Fig 1
). The potential at which current amplitude was maximum in the I-V relationship was -13±1 mV (n=5) for rSkM1 compared with -28.7±3 mV (n=4) for hH1 (Fig 1).
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) and rSkM1 (
). Currents were recorded from -80 to +25 mV in 5-mV increments (holding potential, -100 mV).
Effects of TiTx on rSkM1
In the presence of a concentration as low as 50 nmol/L of TiTx, INa appeared at -70.0±4.0 mV (n=4) (Fig 2A). Thus, the toxin shifted the activation voltage dependence toward more hyperpolarizing potential values. The toxin reached its maximum effect in 5 minutes. The toxin also reduced the peak INa amplitude by 16.6±2.0% (n=5) after 6 minutes. The potential at which current amplitude was maximum on the I-V relationship was not significantly different (-13±1 mV [n=5] without the toxin compared with -15±1 mV [n=5] with the toxin). The t½ value measured at -20 mV was also not significantly different (t½=13.1±0.3 ms [n=5] without the toxin compared with t½=12.9±1 ms [n=5] with the toxin).
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) and without (
Effects of TiTx on hH1
The effects of TiTx on INa due to hH1 were also recorded in cRNA-injected Xenopus laevis oocytes. A concentration of 50 nmol/L TiTx reduced the amplitude of the current of hH1 by 10.9±0.9% (n=4) (Fig 2B
) but did not induce a shift of the activation toward more negative potential values. In order to confirm the absence of the effect of TiTx on hH1 activation, a concentration of 100 nmol/L of toxin was tested on hH1 channels. No effects on activation were observed, and the amplitude of the current was more reduced by 20.8±1.0% (n=3) at 6 minutes. The potential at which current amplitude was maximum on the I-V relationship was not significantly different (-28.7±3 mV [n=4] without the toxin compared with -26±4 mV [n=4] with the toxin). The t½ value measured at -20 mV was also not significantly different (t½=2.6±0.1 ms [n=4] without the toxin compared with t½=2.4±0.1 ms [n=4] with the toxin).
Effects of TiTx on Chimeras Between rSkM1 and hH1
TiTx induces a shift of the activation voltage dependence toward more negative potential values when applied to oocytes injected with cRNA encoding rSkM1 but not when hH1 is being expressed. Since the sensitivity of rSkM1 and hH1 to the effect of TiTx on activation is different, chimeras between these two isoforms were constructed to localize the binding site of TiTx on rSkM1 (Fig 3).
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Effects of TiTx on SSHH
We began by testing the toxin on chimera SSHH, which is composed of D1 and D2 from rSkM1 and D3 and D4 from hH1 (Fig 3). Control INas activate at -46.7±0.7 mV (n=12), whereas in the presence of 50 nmol/L of TiTx, they appear at the more negative voltage of -70.7±1.9 mV (n=7) (Fig 4A). This value is comparable to that obtained with rSkM1. Contrary to the effect of the toxin on rSkM1 and hH1, the amplitude of the current was not reduced by TiTx (n=11). The potential at which current amplitude was maximum on the I-V relationship was not significantly different (-15.7±2 mV [n=7] without the toxin compared with -17.7±2 mV[ n=7] with the toxin). The t½ value measured at -20 mV was also not significantly different (t½=6.1±0.4 ms [n=7] without the toxin compared with t½=6.1±0.8 ms [n=7] with the toxin).
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Effects of TiTx on HSSS
TiTx shifts the activation of SSHH toward more negative potential values. This suggests that the toxin acts on the activation of rSkM1 by binding to D1 and/or D2. In order to extend these findings and clarify between D1 and/or D2, TiTx was tested on chimeras HSSS and SHSS. Chimera HSSS is a channel with D2-D3-D4 from rSkM1 (Fig 3). In the presence of the toxin, a substantial shift of the activation voltage dependence to more negative potentials occurs. Without the toxin, INas activate at -49.0±0.7 mV (n=15), whereas at 50 nmol/L TiTx, they appear at -71.7±1.9 mV (n=12) (Fig 4B). The toxin also reduced the peak INa amplitude by 22.8±2.0% (n=7). The potential at which current amplitude was maximum on the I-V relationship was not significantly different (-22±1 mV [n=12] without the toxin compared with -21±1 mV [n=12] with the toxin). The t½ value measured at -20 mV was also not significantly different (t½=9.6±0.4 ms [n=12] without the toxin compared with t½=9.1±0.5 ms [n=12] with the toxin).
Effects of TiTx on SHSS
As noted above, the toxin causes a shift of the activation voltage dependence of HSSS toward more hyperpolarizing potential. D2 is the most likely candidate for the TiTx interaction region, since HSSS, SSHH, and HSHH have in common only D2 from rSkM1 and all show the phenotype characteristic of rSkM1: HSSS (D3-D4, also from rSkM1), SSHH (D1, also from rSkM1), and HSHH (D1 and D3-D4, from hH1) (Fig 3). Corroboration for this conclusion comes from data on chimera SHSS (D2 from hH1 in a totally rSkM1 background, Fig 3). Except for the small reduction of the amplitude of the current of 7.8±3.0% (n=3) at 6 minutes (Fig 5A), TiTx had no effect on SHSS. In control experiments with SHSS in the absence of toxin, an activation threshold of -42.5±1.0 mV (n=6) was obtained, whereas in the presence of 50 nmol/L TiTx, currents appeared at -41.7±2.4 mV (n=3). The potential at which current amplitude was maximum on the I-V relationship was not significantly different (-17.8±1 mV [n=9] without the toxin compared with -19±1 mV [n=9] with the toxin). The t½ value measured at -20 mV was also not significantly different (t½=4.4±0.2 ms [n=9] without the toxin compared with t½=4±0.1 ms [n=9] with the toxin).
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Effects of TiTx on HSHH
The most compelling proof that TiTx acts on the activation of rSkM1 by interacting with D2 comes from studies of HSHH, a chimera in which only D2 derives from rSkM1 residing in an hH1 background (Fig 3). Although addition of 50 nmol/L TiTx did not induce a reduction in the peak current amplitude (n=9) of HSHH (Fig 5B), as we expected, toxin did produce a shift of the activation toward more negative potential values. In control experiments, INa activates at -49.7±0.9 mV (n=16), whereas in the presence of 50 nmol/L TiTx, INa appears at -68.3±1.7 mV (n=9) (Fig 5B). The potential at which current amplitude was maximum on the I-V relationship was not significantly different (-10±1 mV [n=3] without the toxin compared with -11.6±1 mV [n=3] with the toxin). The t½ value measured at -20 mV was also not significantly different (t½=17.1±1 ms [n=3] without the toxin compared with t½=18.6±1 ms [n=3] with the toxin). These results confirm that toxin interacts with D2 of rSkM1 to modify the activation properties of the voltage-gated NaCh.
Effects of TiTx on rSkM1 Coinjected With ß Subunit
When expressed in Xenopus laevis oocytes, the subunit of rSkM1 has slow inactivation kinetics compared with hH1, which has fast native tissue–like inactivation kinetics. Coinjection of rSkM1s subunit with ß subunit restores the channel's normal fast inactivation kinetics.23 Why rSkM1 has slow inactivation kinetics when expressed in oocytes is not well elucidated.24 Some previous studies have shown that the TiTx slows down the inactivation kinetics of voltage-gated NaChs.14 19 We coinjected ß subunit with subunit encoding for rSkM1 in Xenopus laevis oocytes to restore native tissue–like fast inactivation kinetics in order to evaluate any effect of TiTx on the kinetics of inactivation. Then, we studied the effects of TiTx on rSkM1 subunit coexpressed with ß subunit (Fig 6). The presence of the ß subunit did not modify the effects of TiTx on rSkM1. The threshold of activation was shifted toward more negative values. Without the toxin, inward INas appear at -45.8±1.2 mV (n=12), whereas in the presence of 50 nmol/L TiTx, they activate at -71.0±1.7 mV (n=5). The potential at which current amplitude was maximum on the I-V relationship was not significantly different (-16±2 mV [n=5] without the toxin compared with -19±2 mV [n=5] with the toxin). The t½ value measured at -20 mV was also not significantly different (t½=3.4±0.3 ms [n=5] without the toxin compared with t½=3.3±0.3 ms [n=5] with the toxin).
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Discussion |
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We showed previously that rSkM1 and hH1 have different electrophysiological properties when expressed in the tsA201 cell line22 : differences in threshold of activation, kinetics of inactivation, steady state inactivation, and rate of recovery from inactivation. In the present study, we show that hH1 and rSkM1 also have different patterns of voltage dependence of activation when these two isoforms are expressed in parallel in Xenopus laevis oocytes. The threshold of activation occurs at more hyperpolarizing potentials with hH1. In chimeric channels composed of portions of rSkM1 and hH1, the threshold of activation was found generally to be intermediate between those of hH1 and rSkM1, except for chimera HHSH (with D3 from rSkM1 in an hH1 background). This phenotype of activation threshold seems to be more strongly determined by D1 and D4 (Fig 7
) compared with other domains (with the source of D3 apparently being unimportant within this relatively small series). For example, SSHH, which has two domains from hH1 (D3 and D4) and might have been expected to be more heartlike, activates at more positive potentials (ie, is less hH1-like) than HSSS, which has only a single domain from hH1 (D1) (Fig 7). These data indicate that D1, D2, and D4 play important roles in the activation process by this criterion even though the number of cationic side chains in D3 is greater than that of D2 and D1.
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In neuroblastoma cells,14 TiTx induces (1) a shift of the I-V curve toward more hyperpolarizing potential values and (2) a reduction of the INa amplitude. Our experiments demonstrate an important pharmacological difference between rSkM1 and hH1, voltage-gated NaChs cloned from rat skeletal muscle and human heart muscle, respectively.4 6 When applied to rSkM1, TiTx induces a shift of the activation threshold toward more negative potential values, whereas application to hH1 shows no significant effect of this parameter. In contrast to the suggestion of Vijverberg et al14 that TiTx reveals a population of NaChs that activate at hyperpolarizing potential values, our results show that this toxin induces a shift in the threshold of activation of the -subunit encoding rSkM1 expressed heterologously in Xenopus laevis oocytes. Our observation indicates that this toxin acts by binding to the -subunit of rSkM1. The effect of TiTx on rSkM1 NaChs was found to be concentration dependent (data not shown), being partial at 50 nmol/L. The effect of TiTx was not species dependent, since on the human skeletal muscle NaChs (hSkM1) expressed in Xenopus oocytes, we obtain the same shift in the threshold of activation toward more hyperpolarizing potentials (data not shown). A small reduction of the peak current amplitude for both rSkM1 and hH1 was apparent on exposure to toxin. The use of chimeras between rSkM1 and hH1 permitted localization of the TiTx-induced voltage activation threshold effect to D2 of rSkM1. When D2 of rSkM1 is replaced by the homologous segment of hH1 (chimera SHSS), exposure of this chimera to the toxin has no effect on the activation. Conversely, when D2 of rSkM1 is present (chimeras HSSS, SSHH, and HSHH), the toxin has an effect on activation similar to that observed on rSkM1 (shift toward more negative potential values).
Since the activation process is believed to be related to movement of positive charges on the S4 region toward the outside,25 26 27 the presence or absence of positive charge in the S3-S4 extracellular loop could play a role in activation. The toxin may bind specifically to this region of the channel to alter activation directly by modifying the amount of positive charges on the voltage sensor, or it could be acting indirectly via allosteric mechanisms. There are only three amino acids residues that differ between hH1 and rSkM1 in this region (IIS3-4) (rSkM1/hH1): alanine(654)/serine(799), asparagine(655)/arginine(800), and glycine(658)/asparagine(803). We muted the asparagine(655) residue on rSkM1, and the expressed channel was found to be as sensitive as rSkM1 to TiTx (data not shown); therefore, this amino acid could not account for the difference between hH1 and rSkM1 in terms of their sensitivity to this toxin.
These findings indicate that D2, in addition to D1 and D4 as shown earlier, would play an important role in the activation process of the voltage-gated NaCh. An interesting observation is that the positive charge on the S4 region is not homogeneously distributed among the four domains and that the same pattern of positive charge distribution is found for hH1 and rSkM1. In fact, in both cases, the S4 region of D4 is more positively charged than the S4 region of D3, the S4 region of D3 is more positively charged than that of D2, and the S4 region of D2 is more positively charged than that of D1. These findings indicate that the activation of the NaCh would not only be explained by the simultaneous outward movement of the four S4 regions26 27 but also by the successive movement of the S4 region: first from D4, then from D3, D2, and finally from D1. TiTx could increase the positivity of the S4 region of D2 and thus modify significantly the outward movement of the voltage sensor. An interesting finding to support this hypothesis is that all activation-modifying scorpion toxins (CsE I and CSS II) are positively charged.28
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Acknowledgments |
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This study was supported by the Medical Research Council of Canada MT-12554 (Dr Chahine), the Heart and Stroke Foundation of Canada (Dr Chahine), the American Heart Association, the Muscular Dystrophy Association, the Research Foundation of the University of Pennsylvania, and National Institutes of Health grant AR 41,762 (Dr Kallen). Dr Chahine is Research Scholar of the Heart and Stroke Foundation of Canada. We thank Dr J. Kigma for his comments on the manuscript.
Received June 12, 1996; accepted December 2, 1996.
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References |
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