Introduction

Voltage-gated sodium channels (Navs) are essential for the initiation and propagation of action potentials in excitable tissues such as nerves and muscles1,2,3. They consist of a pore forming α-subunit (260 kDa) associated with auxiliary β-subunits (30–40 kDa)4. In mammals, nine α-subtypes (Nav1.1–1.9) have been identified and cloned5,6. The α-subunit is composed of four homologous domains (DI–DIV) and each domain contains six transmembrane segments (S1–S6). The S5–S6 segments form a central pore for Na+, with the S1–S4 segments from each domain forming the surrounding voltage sensors2,4. Each of the four voltage sensors is activated in response to depolarization of membrane potential7; the voltage sensors of the first three domains (DI–III) are responsible for channel activation, while that of the fourth domain (DIV) determined fast inactivation8,9. Navs fail to inactivate in some cases, resulting in the generation of non-inactivating persistent Na+ currents (INaP), which account for up to 5% of the transient peak inward current (INaT) in physiological conditions10,11. Despite its small amplitude compared with INaT, INaP amplifies synaptic potentials and aids in the repetitive firing of action potentials (AP) in neurons because it is activated by sub-threshold voltages11. The voltage range in which INaP is activated is traversed by interspike intervals during an AP train11,12. INaP has been characterized in a variety of neurons13. Mutations in some Navs genes causing enhanced INaP amplitude are correlated to diseases, such as heart vascular disease and epilepsy11,14,15. Therefore, drugs targeting INaP are expected to have therapeutic benefits16,17,18.

The early understanding of the origin of INaP is based on three hypotheses: (1) the window current hypothesis19; (2) INaP is generated by unusual subtypes of Navs which lacking inactivation20,21; (3) INaP is produced by the same population of channels responsible for INaT through a distinct gating mechanism22,23. Many lines of evidences support the third hypothesis, namely that some channels of a specific Nav subtype occasionally enter the late brief opening and burst of opening state in single channel recordings24,25,26. The single channel mechanism for the slowed inactivation of a Nav by site 3 toxins TsIV-5 and anthopleurin B was also attributed to the increment of mean open time as well as prolonged bursting27,28; studies showed that mutations in all four domains of Navs as well as intracellular loops affect the amplitude of the INaP29; wild type (wt)-Nav1.3 naturally develops INaP in neonatal and axotomized neurons30,31 and Nav1.3 expressed in HEK293T cells displays a clearly detectable INaP32,33. These data also confirm that INaP is the intrinsic property of Navs themselves, although the molecular determinants of INaP in Navs are largely unknown.

In the present study, we identified a tarantula toxin named α-hexatoxin-MrVII (RTX-VII) that enhances the INaP of Nav1.3 and used it as a probe to examine the involvement of each domain of Nav1.3 in the generation of INaP. Our results reveal that domains II and IV work in a synergetic manner to determine the toxin-induced INaP of Nav1.3.

Results

RTX-VII enhances the INaP of Nav1.3

Macrothele raveni (Figure 1a, inset) venom was collected by using an electro-pulse stimulator as described previously34. The lyophilized crude venom was fractionated by RP-HPLC (Figure 1a). A comprehensive screening of each eluted fraction against Nav1.3 transiently expressed in HEK293T cells indicated that the fraction with a retention time of 44.6 min inhibited the fast inactivation of this channel (Figure 1b). This fraction contained a peptide with a molecular weight of 4064.71 Da as determined by MALDI-TOF MS, which was then further purified by analytical RP-HPLC (Supplementary Fig. S1a). Sequence of the peptide was determined by combining Edman degradation (Supplementary Fig. S1j) and cDNA sequencing (Supplementary Fig. S1b) and the toxin was named α-hexatoxin-MrVII (RTX-VII). Blasting the full amino acid sequence of RTX-VII showed that it share 92% identity to a previously known spider toxin Magi-6 (Supplementary Fig. S1c). However, Magi-6 did not compete with the scorpion toxin LqhαIT in binding site 3 of Navs and the symptoms caused by injection of pure Magi-6 to mice could not be directly linked to a particular ion channel receptor35. This raise the possibility that the subtle amino acid sequence variation brought by RTX-VII makes it active on mammalian Navs, as that of APETx3 and APETx1, in which a single amino acid substitution between them confer these two toxins different ion channel selectivity36. RTX-VII contains eight cysteine residues forming four disulfide bonds, as the measured molecular weight was 8 Da less than the theoretical one. The conserved arrangement of the cysteine residues in RTX-VII indicated that it contains a cystine knot (ICK) motif (Supplementary Fig. S1c).

Figure 1
figure 1

RTX-VII induces large INaP in Nav1.3.

(a) RP-HPLC profile of the venom from the spider Macrothele raveni (inset, photo by Dr Songping Liang), where red arrow indicates the fraction containing RTX-VII. (b) Current traces from a representative cell show 0.1 μM RTX-VII enhances the INaT of Nav1.3 and induces a large INaP at the end of a 50-ms depolarization to −10 mV from a holding potential of −100 mV (n = 6). Note INaP was measured at the time point of 45 ms. (c) Time course for the enhancement of INaP of Nav1.3 by 0.2 μM RTX-VII and the recovery upon washing with bath solution, τon and τoff is 40.9 ± 11.3 s and 162.8 ± 39.7 s, respectively (n = 4). (d) RTX-VII dose dependently enhances INaP of Nav1.3 with an apparent EC50 of 0.12 μM (n = 6). The maximum response (fmax) and the minimum response (fmin) of Nav1.3 to RTX-VII is 66.90% and 0, respectively.

As shown in Figure 1b, RTX-VII had three effects on current of Nav1.3: (1) it increased the INaT amplitude at the depolarizing voltage of −10 mV; (2) it inhibited the fast inactivation of the channel as determined by I5ms/INaT ratio; (3) and it induced a large INaP as revealed by the I45ms/INaT ratio. At a depolarization of −10 mV, the INaP generated by Nav1.3 accounted for little of the INaT under control conditions, whereas the treatment with 0.1 μM RTX-VII enhanced the INaP to approximate 25% of the INaT. The INaP evoked by the toxin lasted for several seconds and large tail current was observed when cell membrane was repolarized (Supplementary Fig. S1d), distinguishing this toxin from certain α-scorpion toxins. The time course for 0.2 μM RTX-VII activating the INaP of Nav1.3 was characterized by a slow onset of action (τon = 40.9 ± 11.3 s) and a slow recovery upon washing (τoff = 162.8 ± 39.7 s) (Figure 1c). The activation of the INaP of Nav1.3 by RTV-VII was dose-dependent, with an apparent EC50 of 120 nM (Figure 1d). The activity and selectivity of RTX-VII were examined against three Nav subtypes (Nav1.4, 1.5 and 1.7) expressed in HEK293T cells, TTX-R Navs of rat dorsal root ganglion neurons and Navs in neonatal rat hippocampal neurons. Among these channels, Navs of neonatal rat hippocampal neurons were sensitive to this toxin (Supplementary Fig. S1i), whereas the others were not (Supplementary Fig. S1e–h).

Kinetics of RTX-VII action on Nav1.3

The current-voltage (I–V) relationships of the INaT and INaP of Nav1.3 before and after the application of 0.2 μM RTX-VII were explored (Figure 2, Supplementary Fig. S2). Compared with the control, RTX-VII modified the I–V relationship of the INaT as follows: (1) the activation of the INaT was potentiated by the toxin at voltages ranging from −45 mV to 10 mV, while no potentiation was observed at voltages > 10 mV (Figure 2a); (2) the activation voltage of the maximum INaT was shifted from 5 mV in the control to −10 mV in the presence of the toxin (Figure 2a, Supplementary Fig. S2d). Although RTX-VII did not alter the reversal voltages (approximate 65 mV) of Nav1.3, toxin application did negatively shift channels' initial activation voltage (Figure 2a and c). These data indicate that the toxin treatment may increase the opening probability of Nav1.3 channels in cell membrane and facilitate their activation at weak depolarizing voltages. The I–V curves of the INaP before and after the application of toxin indicate that the enhancement of the INaP by the toxin occurred across the depolarizing voltages tested (Figure 2a, Supplementary Fig. S2a) and the activation voltage of the maximum INaP was at about −10 mV. Regarding the I–V curves of the INaP and INaT in the presence of toxin, if the amplitudes of the INaP and INaT at each depolarizing voltage were normalized to their maximum one, respectively, they overlapped completely (Figure 2b), suggesting that the INaP and INaT in the presence of the toxin share rather similar activation voltage. Similar to some α-scorpion toxins37, RTX-VII removed the fast inactivation of Nav1.3 in a voltage-independent way at depolarizing voltages ranging from −20 mV to +30 mV (Supplementary Fig S2b).

Figure 2
figure 2

Kinetics for RTX-VII interacting with Nav1.3.

(a) I–V curves of the INaP and INaT of Nav1.3 before and after 0.2 μM RTX-VII treatment. Each current component was normalized to the maximum INaT in control (n = 11). (b) I-V curves of the INaT and INaP of Nav1.3 in the presence of 0.2 μM RTX-VII. The INaP and INaT at each depolarizing voltage were normalized to their maximum one, respectively (n = 11). (c) A cluster of current traces from a representative cell show that 0.2 μM RTX-VII enhances the INaT and INaP of Nav1.3 at various depolarizing voltages (n = 11). (d) 0.2 μM RTX-VII causes a hyperpolarized shift of the steady-state activation curve of Nav1.3 without changing the slope factor (Va = −12.72 ± 4.92 mV for control and Va = −29.64 ± 6.21 mV for the toxin treatment; Ka = 6.83 ± 0.98 mV for control and Ka = 5.66 ± 1.12 mV for the toxin treatment) (n = 11), as well as a small but significant hyperpolarized shift of the steady-state inactivation accompanied by a significant change of the slope factor (Vh = −44.58 ± 2.73 mV for control and Vh = −51.12 ± 5.00 for the toxin treatment, p < 0.05; Ka = −6.98 ± 1.44 mV for control and Ka = −12.49 ± 0.41 mV for the toxin treatment, p < 0.001) (n = 5). (e) Representative traces shows the recovery of Nav1.3 from fast inactivation before (control) and after (toxin) 0.2 μM RTX-VII treatment. The red arrow indicates the recovery duration of 0 ms; the numbers labeled above the traces show the recovery time. The repriming protocol is also shown (n = 5). (f) Time-dependent recovery of Nav1.3 from fast inactivation in the presence and absence of 0.2 μM RTX-VII (n = 5).

The conductance-voltage (G–V) relationship and the steady-state inactivation of Nav1.3 before and after the application of RTX-VII were explored (Figure 2d). Compared with the control, RTX-VII increased the conductance of the cell membrane at depolarizing voltages below 10 mV as revealed by an approximate 16 mV negative shift of Nav1.3 channels' activation curve induced by the toxin (Va = −12.72 ± 4.92 mV for control and Va = −29.64 ± 6.21 mV for the toxin treatment), which is in accordance with the negative shift of the activation voltage for maximum INaT observed in the I–V curve; the toxin did not significantly alter the slope factor of the activation curve (Ka = 6.83 ± 0.98 mV for control and Ka = 5.66 ± 1.12 mV for the toxin treatment). The I–V and G–V relationships of Nav1.3 before and after RTX-VII application were acquired with stringent controls of the uncompensated series resistance (Rs) caused depolarizing voltage error (the maximum tolerable voltage error was less than 5 mV, the mean maximum Rs-caused voltage error was 2.34 ± 1.08 mV, p < 0.001 when compared to the Va shifted amplitude). A steady-state component (approximate 20% of the INaT) that was resistant to inactivation was observed in the steady-state inactivation (SSI) curve when conditional voltages were above −20 mV, which should represent the INaP elicited by conditional pulses. A significant change of Vh and Kh were observed (Vh = −44.58 ± 2.73 mV for control and Vh = −51.12 ± 5.00 mV for the toxin treatment, p < 0.05; Kh = −6.98 ± 1.44 mV for control and Kh = −12.49 ± 0.41 mV for the toxin treatment, p < 0.001). The hyperpolarization shift of the G–V curve and a non-inactivated component in the SSI curve together resulted in an enlarged voltage range for generation of window current, indicating a slower development of closed state inactivation (CSI) in the toxin-treated channels.

The effect of RTX-VII on the repriming kinetics (recovery from fast inactivation) of Nav1.3 was also investigated. As shown in Figure 2e, the INaT of Nav1.3 recovered gradually from fast inactivation with the repolarizing time (recovery time) increasing in the absence (control) and presence (toxin) of RTX-VII. The INaP induced by the toxin was observed at all recovery time. The INaP of the toxin-treated channels fully recovered at the recovery time of 0 ms, but no INaT recovery was observed (Figure 2e, toxin). The recovery ratios of Nav1.3 INaT before and after the application of toxin were plotted as a function of recovery time (Figure 2f), showing most of channels (>80%) recovered from fast inactivation in 4 ms in both conditions. An apparently faster repriming of the toxin-treated channels than that of control channels within 4 ms was observed, which could be associated with the existence of the INaP. If the INaP was subtracted from the INaT in toxin treated channels, the residual current would exhibit the same repriming kinetics as that of the control (Supplementary Fig. S2c).

The molecular mechanism of RTX-VII as an excitatory toxin

The enhancement of INaP of Navs in hippocampal neurons by RTX-VII may have led to excitatory toxic in mouse. Intracerebroventricular injection of 20 ng RTX-VII dissolved in 20 μl saline caused seizure-like symptoms, as described by circular running in the first several minutes followed by involuntary body twitching, while animals in control group injected with 20 μl saline behaves normal (n = 5 in each group, Supplementary video). We therefore investigated the mechanism of RTX-VII as excitatory toxin. Nav1.3 is upregulated in the peripheral nervous system in response to nerve injury and contributes to the hyperexcitability of nociceptive neurons under neuropathic conditions38,39. The fast repriming kinetics and slow development of CSI of Nav1.3 make it suitable for generating a large response to slowly developing depolarizing inputs (ramp stimuli)40. We first tested the effect of RTX-VII on the ramp current (Iramp) of Nav1.3 evoked by various ramp stimuli (Figure 3a p1). Consistent with previous studies33, Nav1.3 expressed in HEK293T cells produced a large inward Na+ current in response to a linearly increasing voltage ramp from −100 mV to 20 mV at the ramp rate of 1.2 mV/ms; of the two Iramp peaks shown, the first one (Iramp1) but not the second one (Iramp2) was ramp rate-dependent, with higher rate leading to larger Iramp1 (Figure 3b, black trace). The application of 0.5 μM RTX-VII increased the amplitude of both Iramp1 and Iramp2 generated by Nav1.3 at all ramp rates tested along with a hyperpolarization shift of the initial activation voltage for Iramp1 (Figure 3b, red trace; the maximum tolerable voltage error was less than 5 mV, the mean maximum Rs-caused depolarizing voltage error was 3.45 ± 1.26 mV).The negative shift of Iramp1 of Nav1.3 was consistent with the channels' negatively shifted activation. The enhanced activation of Iramp1 of Nav1.3 may have been derived from the larger potential gradient (caused by the negative shift of activation voltage of Iramp1) that drives Na+ to cross the membrane as well as a slowed CSI which makes more channels available for activation.

Figure 3
figure 3

The effects of RTX-VII on the ramp currents of Nav1.3 and Navs of hippocampal neurons.

(a) Protocols are used in the experiments described in this figure. (b) A series of inward Nav1.3 currents are evoked by using linearly increasing ramp voltage from −100 mV to 20 mV with different ramp rate (Figure 3a, p1), the ramp time ranges from 100 ms to 600 ms, 100 ms/step. The Nav1.3 ramp current (Iramp) displays two peaks with the first one (Iramp1) but not the second one (Iramp2) being sensitive to ramp rate in control (black traces). 0.5 μM RTX-VII enhances the amplitudes of both peaks and causes a hyperpolarized shift of the initial activation voltage for Iramp1 (red traces); numbers labeled above the traces indicate the ramp rate (mV/ms) (n = 10). (c) Representative traces show that RTX-VII dose-dependently enhances the INaP (I45ms) and Iramp2 of Nav1.3 elicited by the protocol p2 shown in Figure 3a (n = 5). (d) Dose-response curve for RTX-VII activating the Iramp2 of Nav1.3, the apparent EC50 is determined as approximate 0.3 μM; the maximum and the minimum response of Nav1.3 to RTX-II is 63.03% and 2.15%, respectively (n = 5). (e) The I45ms/INaT ratio was plotted as a function of the Iramp2/INaT ratio at each toxin concentration (data from Figure 3c). A linear fit of the dots shows the close correlation between the INaP and Iramp2 of Nav1.3 (R2 = 0.9947) (n = 5). (f) Compared with control, 1 μM RTX-VII evidently enhances both peaks (Iramp1 and Iramp2) of the ramp current of Navs in rat hippocampal neurons (upper). Protocol p3 shown in Figure 3a was used (n = 5); Representative traces (below) show that the Iramp of Navs in hippocampal neurons is elicited by protocol p4 shown in Figure 3a (n = 5). (g) Spontaneous AP firing in a neonatal rat hippocampal neurons in the absence (below) and presence (upper) of 2 μM RTX-VII (n = 8).

Nav1.3 intrinsically produces small INaP and the relationship between INaP and Iramp2 was investigated in a previous study in which a close correlation between them was observed33. To clarify the relationship between the RTX-VII evoked INaP and Iramp2, the protocol p2 described in Figure 3a was used to elicit two type currents of Nav1.3 (Figure 3c). Note only Iramp2 could be evoked at the ramp rate of 0.2 mV/ms (Figure 3b and Figure 3c). RTX-VII dose-dependently enhanced INaP (I45ms) and Iramp2 of Nav1.3 (Figure 3c).The apparent EC50 for RTX-VII activating Iramp2 was 320 nM, as revealed by plotting Iramp2/INaT ratios as a function of toxin concentrations (Figure 3d). This EC50 value did not differ much from that of RTX-VII activating the INaP of Nav1.3 (120 nM). Furthermore, the correlation coefficient between INaP and Iramp2 was 0.9947 (Figure 3e), indicating a close correlation between them. Thus, data derived from the toxin study further confirmed the conclusion described above.

The effect of RTX-VII on the ramp current of Navs in neonatal hippocampal neurons was also examined. As shown in Figure 3f, both Iramp1 and Iramp2 of hippocampal Navs were evoked by a linearly increasing voltage ramp from −100 mV to 20 mV at the ramp rate of 1.2 mV/ms (Figure 3a, p3); both components of the Iramp of hippocampal Navs were greatly enhanced by toxin (Figure 3f, upper). As shown in Figure 3f (below), both Iramp1 and Iramp2 of hippocampal Navs in control conditions displayed voltage-dependent inactivation by reverse ramp (R-ramp) stimulation following forward ramp (F-ramp) stimulation (Figure 3a, p4); as Iramp1 disappeared, the amplitude of Iramp2 decreased in the R-ramp compared with that in the F-ramp. On the contrary, 1 μM RTX-VII treatment removed the voltage-dependent inactivation of Iramp2 but not Iramp1, as toxin induced a nearly unchanged Iramp2 in both the forward and reverse ramps, while Iramp1 was absent in the R-ramp. This finding indicates that the amplitude of Iramp2 in the presence of the toxin is only dependent on the transmembrane potential and this population of Navs generating Iramp2 should maintain a continuous open state during the entire time course of AP. The toxin negatively shifted Iramp1 and the enhanced activation of Iramp2 might lower the threshold and increase the frequency of AP in hippocampal neurons, respectively, which possibly triggers the spontaneous AP firing in hippocampal neurons at a physiological resting potential. Current-clamp experiments showed that 2 μM RTX-VII triggered spontaneous high frequency AP firing in hippocampal neurons (Figure 3g, upper; Supplementary Fig. S3), a mechanism underlying toxin-induced seizure-like symptom in mice. By contrast, the spontaneous AP firing was rare in hippocampal neurons under control conditions (Figure 3g, below; Supplementary Fig. S3).

Domains II and IV of Nav1.3 are critical for INaP generation by RTX-VII

Because Nav1.5 is resistant to RTX-VII (Figure 4b), a chimera strategy was used to screen the critical modules [voltage sensor domains (VSD) or pore domains (PD)] responsible for the toxin-induced INaP of Nav1.3. Each module from the four domains of Nav1.3 was substituted with the corresponding Nav1.5 module (Supplementary Fig. S4). The nomenclature of a specific chimeric channel was defined as follows: for example, Nav1.3/1.5 DI-VSD chimera is a hybrid channel in which the DI-VSD of Nav1.3 was replaced with that of Nav1.5. Eight Nav1.3 derived chimeric channels were constructed. All chimeric channels except the Nav1.3/1.5 DII-VSD chimera were functionally expressed in HEK293T cells; therefore, the hybrid channel Nav1.3/1.5 DII was generated instead of the Nav1.3/1.5 DII-VSD chimera (Supplementary Fig. S4). To assess the potency and efficacy of RTX-VII for INaP generation in each wt- or chimeric channel, a 300-ms depolarization to 10 mV from a holding potential of −100 mV was applied to evoke the INaT and INaP of a specific channel in the absence and presence of various concentrations of toxin and the INaP was measured at the time point of 295 ms (Figure 4a) because the currents of chimeric channels reached a macroscopic steady state at the time point of 300 ms. To compare data derived from different channels, the relative values of INaP/INaT (both from the same current trace) after treatment with different concentrations of toxin were calculated and the potency of RTX-VII on a specific channel was defined as the EC50 value, while the efficacy of RTX-VII was determined by steady-state INaP/INaT ratio at the saturated concentration of toxin.

Figure 4
figure 4

Domain II and IV of Nav1.3 are synergistically involved in INaP generation.

(a) A 300-ms recording of currents of wt-Nav1.3 and Nav1.3 derived chimeric channels before and after the application of various concentration of RTX-VII. Chimeric channels were constructed as follows: the voltage-senor domain (VSD) or pore domain (PD) of DI, DII, DIII or DIV of Nav1.3 was substituted with the corresponding domain of Nav1.5 (see Supplementary Fig. S4). Here, INaP was measured at time point of 295 ms. Note DII, DIV-VSD, DIV-PD substitutions in Nav1.3 results in the reduction of toxin induced INaP compared with wt- and other chimeric channels (n = 7–11). (b) Representative traces show that Nav1.5 is resistant to RTX-VII (n = 4). (c) Dose-response curves for RTX-VII activating the INaP of wt-Nav1.3 channel and Nav1.3 derived chimeric channels that did not or slightly changed toxin potency (EC50) or efficacy (steady-state INaP/INaT ratio at the saturated concentration of the toxin) (n = 7–11). (d) Dose-response curves for RTX-VII activating the INaP of wt-Nav1.3 and chimeric channels that dramatically changed toxin potency and/or efficacy (n = 7–11). (e) Bars show the fold changes of the apparent EC50 of RTX-VII on each Nav1.3 derived chimeric channel compared with that for wt-Nav1.3 (n = 7–11). (f) Bars show ratios of the steady-state INaP/INaT of wt- and Nav1.3 derived chimeric channels in the presence of the saturated concentration of toxin. These values are 45.88 ± 4.78%, 52.78 ± 8.32%, 48.87 ± 6.09%, 24.46 ± 5.27%, 40.64 ± 5.47%, 53.95 ± 8.13%, 49.00 ± 5.88%, 7.16 ± 2.33% and 24.48 ± 7.52% for the chimeric channels Nav1.3, 1.3/1.5 DI-VSD, 1.3/1.5 DI-PD, 1.3/1.5 DII, 1.3/1.5 DII-PD, 1.3/1.5 DIII-VSD, 1.3/1.5 DIII-PD, 1.3/1.5 DIV-VSD and 1.3/1.5 DIV-PD, respectively (***p < 0.001, when compared with wt-1.3) (n = 7–11).

The substitution of the VSD/PD of Nav1.3 with that of Nav1.5 had different effects on the potency and efficacy of RTX-VII. Compared with the wt-channel, five chimeric channels, namely Nav1.3/1.5 DI-VSD, DI-PD, DII-PD, DIII-VSD and DIII-PD produced a large INaP in response to RTX-VII, whereas the other three chimeric channels, Nav1.3/1.5 DII, DIV-VSD and DIV-PD displayed a smaller INaP (Figure 4a). The apparent EC50 values of RTX-VII on these channels were further determined from dose-response curves (Figure 4c and d). The bar diagrams shown in Figure 4e and f indicate the changes in the potency and efficacy of RTX-VII on Nav1.3 derived chimeric channels, respectively, which could be described as follows: (1) in the chimeras Nav1.3/1.5 DI-VSD, DII-PD, DIII-VSD and DIII-PD, no significant changes in the potency and efficacy of RTX-VII were observed, as indicated by the negligible reduction in the steady-state INaP/INaT ratios and the less than two-fold increments of EC50 values; (2) the chimera Nav1.3/1.5 DI-PD generated a large steady-state INaP, similar to that of the wt-Nav1.3; however, an approximate 4.8 fold increase of the EC50 value was observed, indicating that this chimeric channel reduced the binding affinity of RTX-VII but not the efficacy; (3) the chimeras Nav1.3/1.5 DII and DIV-PD significantly decreased the efficacy of RTX-VII, as revealed by a significantly smaller steady-state INaP/INaT ratios (steady-state INaP only accounts for approximate 24% of INaT after 2 μM RTX-VII treatment, P < 0.001 when compared to wt-Nav1.3); however, these substitution resulted in a < 2-fold increase of the EC50 values; (4) In the chimera Nav1.3/1.5 DIV-VSD, both the potency and efficacy of RTX-VII was significantly attenuated, because the toxin, even at a concentration of 10 μM, induced a small fraction of steady state INaP (<10% of the INaT, P < 0.001, when compared to wt-Nav1.3) in this chimeric channel and the apparent EC50 of toxin on this chimeric channel was increased by approximate 25 folds when compared to wt-Nav1.3. Taken together, these findings suggest that DIV-VSD, DIV-PD, DII and DI-PD of Nav1.3 play important roles in the RTX-VII-induced INaP. The different influence of these Nav1.3 module substitutions on the potency and efficacy of RTX-VII suggest that they play different roles. DIV-VSD and DI-PD might jointly compose the binding receptor for RTX-VII, whereas the loss of efficacy of RTX-VII on the chimeric channel Nav1.3/1.5 DII was not caused by loss of toxin binding but was rather associated with an intrinsic limitation of this hybrid channel in generating a larger INaP.

Domain II of Nav1.3 is not involved in interacting with RTX-VII

Further experiments were performed to clarify the roles of Nav1.3 DII and DIV. First, we examined whether RTX-VII binds to Nav1.3 DII. Neurotoxins acting on DII of Navs often cause a negative or positive shift of the activation kinetics of targeted channels41. The substitution of the DII of Nav1.3 with that of Nav1.5 should affect the RTX-VII-induced negative shift of activation kinetics of Nav1.3 if the toxin binds to Nav1.3 DII, as RTX-VII did not affect the I–V curve of Nav1.5 (Supplementary Fig. S5a). Therefore, the activation kinetics of the chimeric channel Nav1.3/1.5 DII was investigated before and after the application of 2 μM RTX-VII. RTX-VII negatively shifted the voltage-dependent activation of the Nav1.3/1.5 DII chimera and increased the INaT at voltages ranging from −50 mV to 5 mV (Figure 5a). In addition, 2 μM RTX-VIII caused an approximate 14 mV negative shift of the G–V curve of the Nav1.3/1.5 DII chimera without changing the slope factor (Va = −18.00 ± 1.71 mV for control and Va = −32.09 ± 1.99 mV for the toxin treatment; Ka = 7.23 ± 1.22 mV for control and Ka = 6.82 ± 1.30 mV for the toxin treatment; the maximum tolerable voltage error was less than 5 mV, the mean maximum Rs-caused depolarizing voltage error was 2.92 ± 2.10 mV, p < 0.001 when compared to the Va shifted amplitude) (Figure 5b).This raises the possibility that the toxin might not interact with Nav1.3 DII, which was further confirmed by using a competitive assay. HNTX-III is a tarantula toxin that inhibits the INaT of Nav1.3 and Nav1.7. It was found that this toxin targeted DII S3–S4 linker of Nav1.742. The wt-Nav1.5 channel and the Nav1.3/1.5 DII chimera were resistant to 1 μM HNTX-III treatment, whereas the Nav1.3/1.5 DII-PD chimera was inhibited by 1 μM HNTX-III (Supplementary Fig. S5 b–d). Reconstruction of the DII of Nav1.3 to Nav1.5 (Nav 1.5/1.3 DII chimera) conferred the inhibitory activity of HNTX-III to this channel (Supplementary Fig. S5 e).These evidences indicate HNTX-III inhibit Nav1.3 by binding to its DII-VSD. If RTX-VII also targeted DII-VSD of Nav1.3, its binding should prevent the interaction of HNTX-III with Nav1.3 because of steric hindrance, which would result in an attenuation of the inhibitory potency of HNTX-III on Nav1.3. As shown in Figure 5c, the inhibitory effects of HNTX-III on Nav1.3 INaT did not differ between 0.5 μM RTX-VIII-pretreated and -untreated channels. The dose-response curves were also superimposed well (Figure 5d), providing evidence to rule out the binding of RTX-VII to Nav1.3 DII. Next, we determined the molecular determinant in DIV of Nav1.3 for RTX-VII binding. Since Nav1.5 is resistant to RTX-VII, the residues in S1–S2 and S3–S4 extracellular loops of Nav1.3 were mutated to the corresponding residues of Nav1.5, respectively (Figure 5e). A total of seven residues were mutated and six of them were functionally expressed except V1566F. The kinetics for the activation and SSI of all mutants were listed in supplementary Table S1. Compared with wt-Nav1.3, Four mutant channels (K1503P, M1505K, T1506I, L1507N) carrying mutations in the S1–S2 linker led to a 4–12 folds increase of apparent EC50 values, whereas the E1562Q and E1562R mutation in the S3–S4 linker resulted in an approximate 5 folds and 20 folds increase of the apparent EC50 values (Figure 5f and 5g). These data indicate that multiple residues located in Nav1.3 DIV were involved in interacting with RTX-VII and that E1562 was the most important residue for the interaction.

Figure 5
figure 5

Domain IV but not domain II of Nav1.3 is involved in interacting with RTX-VII.

(a) The I–V curves of Nav1.3/1.5 DII chimera before and after 2 μM RTX-VII treatment (n = 6). (b) 2 μM RTX-VII negatively shifts the G–V curve of Nav1.3/1.5 DII chimera without changing the slope factor (Va = −18.00 ± 1.71 mV for control and Va = −32.09 ± 1.99 mV for the toxin treatment; Ka = 7.23 ± 1.22 mV for control and Ka = 6.82 ± 1.30 mV for the toxin treatment) (n = 6). (c) Representative traces show 1 μM HNTX-III indiscriminately inhibits the INaT of 0.5 μM RTX-VII-untreated- (RTX-VII free) and -treated- (RTX-VII pretreated) Nav1.3 channel (n = 5). HNTX-III was dissolved in bath solution containing 0.5 μM RTX-VII. (d) The dose-response curve for HNTX-III inhibiting the INaT of 0.5 μM RTX-VII-treated or -untreated Nav1.3 channel shows that the potency of HNTX-III on both types of channel are the same (n = 5). The fractions which are resistant to the high dose of HNTX-III treatment account for 5.12% and 6.77% of the maximum INaT, respectively. (e) Sequence alignment of the DIV-VSD of Nav1.3 and Nav1.5, red arrows indicate amino acid residues in Nav1.3 which were mutated to their counterpart in Nav1.5. (f) Dose-response curves for RTX-VII enhancing the INaP of Nav1.3 mutants shown in Figure 5e demonstrate molecular determinants in Nav1.3 for interacting with RTX-VII (n = 5–8); the apparent EC50 values are 117.73 nM, 741.48 nM, 208.83 nM, 541.00 nM, 1209.76 nM, 1399.27 nM, 635.04 nM, 2451, 32 nM for wt-Nav1.3, K1503P, Y1504E, M1505K, T1506I, L1507N, E1562Q and E1562R, respectively. (g) Bars show the fold changes of apparent EC50 values of RTX-VII for mutants compared with that for wt-Nav1.3 channel (n = 5–8).

Reverse reconstruction of Nav1.3 DII and DIV into Nav1.5 fully restores toxin efficacy

Considering the critical role of the DII and DIV of Nav1.3 in the RTX-VII-induced INaP, we assumed that reverse reconstruction of Nav1.3 DII and DIV into Nav1.5 might restore the efficacy of the toxin. A reversal chimeric strategy was used as follows: four domains of Nav1.3 were stepwise reconstructed into the scaffold of Nav1.5 (Supplementary Fig. S6). The nomenclature of a chimeric channel was defined as follows: for example, Nav1.5/1.3 DI was a chimeric channel in which the DI of Nav1.5 was substituted with that of Nav1.3. A total of 11 chimeric channels were constructed and their INaP generation by the toxin was compared. Again, INaP was measured at the time point of 295 ms (Figure 6a). The substitution of all four domains of Nav1.5 with those of Nav1.3 (Nav1.5/1.3 DI-II-III-IV) almost fully restored the efficacy of RTX-VII, thus eliminating the involvements of the intracellular loops of Nav1.3 in the toxin-induced INaP. Of the four single domain replaced chimeric channels, Nav1.5/1.3 DI, Nav1.5/1.3 DII and Nav1.5/1.3 DIII chimeras were resistant to RTX-VII, similar to wt-Nav1.5, whereas Nav1.5/1.3 DIV chimera was sensitive to RTX-VII. Furthermore, the toxin slowed the inactivation and induced a small steady- state INaP in this chimeric channel, indicating that Nav1.3 DIV is important but not sufficient for RTX-VII inducing large INaP. Of the two triple domain replaced chimeric channels, Nav1.5/1.3 DI-III-IV chimera did not fully restore toxin efficacy but Nav1.5/1.3 DI-II-IV chimera did, which indicates that the DII but not the DI and DIII of Nav1.3 is required for toxin inducing large INaP. Of the three double domain replaced chimeric channels, the reconstruction of the DI or DIII of Nav1.3 into the scaffold of Nav1.5/1.3 DIV chimera (Nav1.5/1.3 DIII-IV chimera or Nav1.5/1.3 DI-IV chimera) had a limited effect on restoring toxin efficacy, whereas the reconstruction of the DII of Nav1.3 into Nav1.5/1.3 DIV chimera (Nav1.5/1.3 DII-IV chimera) almost fully rescued toxin efficacy, suggesting the assembly of the DII and DIV of Nav1.3 should be sufficient for RTX-VII inducing large INaP. Additionally, the chimeric channel Nav1.5/1.3 DI-III-IV&DII PD, where only the DII-PD but not the whole DII of Nav1.3 was present, also attenuated the efficacy of RTX-VII compared with that of Nav1.5/1.3 DI-II-III-IV chimera, which strongly supports that the DII-VSD of Nav1.3 plays a vital role in toxin-induced INaP generation.

Figure 6
figure 6

The substitution of the domain II and IV of Nav1.5 with those of Nav1.3 restores RTX-VII efficacy.

(a) A 300-ms recording of the currents of Nav1.5 derived chimeric channels in the absence and presence of various concentration of RTX-VII (n = 6–9). The chimeric channels were constructed as follows: one or several domains (DI, DII, DIII or DIV) of Nav1.5 were substituted with the corresponding domain/s of Nav1.3 (see Supplementary Fig. S6). (b) Dose-response curves for RTX-VII enhancing the INaP of wt-Nav1.3 and Nav1.5 derived chimeric channels that did not or slightly restored toxin efficacy (steady-state INaP/INaT ratio at the saturated concentration of toxin) (n = 6–9). (c) Dose-response curves for RTX-VII enhancing INaP of wt-Nav1.3 and Nav1.5 derived chimeric channels that almost completely restored toxin potency and/or efficacy (n = 6–9). (d) Bars show the fold changes of the apparent EC50 of RTX-VII for each Nav1.5 derived chimeric channels compared with that for wt-Nav1.3 (n = 6–9). (e) Bars show the steady- state INaP/INaT ratio of wt-Nav1.3 and Nav1.5 derived chimeric channels in the presence of saturated concentrations of toxin. This values are 47.92 ± 5.43%, 46.37 ± 8.87%, 6.3 ± 1.82%, 10.60 ± 3.32%, 53.81 ± 5.56%, 12.68 ± 3.67%, 50.88 ± 8.33 and 20.41 ± 3.90% for Nav1.3, 1.5/1.3 DI-DII-DIII-DIV, 1.5/1.3 DIV, 1.5/1.3 DI-DIII-DIV, 1.5/1.3 DI-DII-DIV, 1.5/1.3 DI-DIV, 1.5/1.3 DII-DIV and 1.5/1.3 DI-DIII-DIV&DII-PD, respectively (***p < 0.001, N.S = not significant, when compared with wt-Nav1.3) (n = 6–9).

The apparent EC50 values of RTX-VII on the Nav1.5 derived chimeric channels containing Nav1.3 DIV were estimated from the dose-response curves (Figures 6b and c) and the changes in the potency and efficacy of RTX-VII on these chimeric channels were showed in Figures 6d and e, respectively. Of the Nav1.3 DIV-containing chimeric channels, the Nav1.3 DII-containing ones (Nav1.5/1.3 DI-II-III-IV, DI-II-IV and DII-IV chimeras), but not those without reconstruction of Nav1.3 DII or DII-VSD (Nav1.5/1.3 DIV, DI-III-IV, DI-IV and DI-III-IV&II-PD chimeras), produced a large steady-state INaP comparable to that of wt-Nav1.3 in the presence of saturated concentration toxin (Figure 6e). On the other hand, the toxin potency on these Nav1.3 DIV-containing chimeric channels were only slightly weaker than that of wt-Nav1.3, although the greatest fold change of EC50 was observed in Nav1.5/1.3 DII-DIV chimera (8 folds) (Figure 6d). Moreover, the incorporation of Nav1.3 DI into Nav1.5/1.3 DII-DIV chimera (Nav1.5/1.3 DI-DII-DIV chimera) led to an evident enhancement of toxin potency, which is comparable to that of wt-Nav1.3 channel. The results are consistent with the interpretation that the DIV of Nav1.3 was the main toxin binding site, while the DI-PD of Nav1.3 might construct the low affinity binding site for RTX-VII. Overall, combining data in Figures 4 and 6 confirmed the cooperative involvement of DII and DIV in the toxin-induced INaP of Nav1.3.

Discussion

Neurotoxins produced by venomous animals, plants and microorganisms are a valuable pool of molecular probes to investigate the structure-function relationship of Navs43. RTX-VII robustly enhances the INaP of Nav1.3 and discriminates Nav subtypes Nav1.4, Nav1.5 and Nav1.7–1.9 from Nav1.3. The toxin-induced and the intrinsic INaP share some common features, such as sub-threshold activation, a close correlation with Iramp2 and triggering spontaneous high frequency AP firing. Furthermore, the brief late opening and burst of openings of Navs may be the common mechanism underlying the origin of both types of INaP. However, the intrinsic INaP of Navs is small, which hampered the investigation of the mechanism underlying INaP generation. RTX-VII dramatically enhancing the INaP of Nav1.3 enabled detailed investigations of Nav1.3INaP generation. In the present study, we clarified the roles of the four domains of Nav1.3 in INaP generation by using RTX-VII as a molecular probe.

Along with the enhancement of INaP, RTX-VII also facilitates Nav1.3 channel opening at weak depolarizations as revealed by the toxin potentiating INaT of Nav1.3 when depolarizing voltages are below 10 mV as well as the toxin negatively shifting channel's steady-state activation. This observation is not without precedent, as some α-scorpion toxins modulate Navs in a similar way37. This phenomenon could be reasonably interpreted as an increase of the maximum opening probability of the toxin-treated channels. However, how the toxin-bound channels open with a greater probability at weak depolarizations remains unclear. Our data indicate that RTX-VII binds to the DIV-VSD instead of the DII-VSD of Nav1.3, which suggests that the potentiation of Nav1.3 activation by the toxin might not derive from the toxin facilitating DII activation. Two possible explanations for RTX-VII enhancing the INaT of Nav1.3 are proposed: (1) toxin treatment altered single channel conductance44 of Nav1.3. This interpretation seems unreasonable because the toxin does not alter the inward and outward Na+ current of Nav1.3 evoked by strong depolarizing voltages above 10 mV; (2) RTX-VII tends to stabilize the DIV-VSD of Nav1.3 in a partially activated state (pre-activated state), which is required for channel activation but not sufficient to trigger channel inactivation (the fully activated DIV-VSD is required for the fast inactivation of Navs)45, thus promoting channel activation in a similar but not identical way with that of β-scorpion toxins46,47. The vital difference is that β-scorpion toxins trap the DII-VSD but not DIV-VSD of Navs in the activated state. The latter interpretation seems plausible as emerging evidences support DIV is involved in Nav activation45,48. Furthermore, the voltage driving the outward movement of DIV-VSD is the later step in the activation sequence of Navs49. RTX-VII did not alter the INaT of Nav1.3 at strong depolarizing voltages (>10 mV) and this phenomenon could be interpreted by the fact that both toxin-free and toxin-bound channels in cell membrane are almost fully activated at 10 mV, which is in consistent with the G-V relationship observed in figure 2d. Taken in all, considering RTX-VII promoting activation and inhibiting inactivation of Nav1.3 as well as the unique role of DIV-VSD in channel gating, we would like to suggest that RTX-VII might tend to trap and stabilize the DIV-VSD of Nav1.3 in the pre-activated state during channel activation.

The reconstruction of Nav1.3 DII but not DI or DIII to Nav1.5/1.3 DIV chimera fully restored toxin efficacy, but it is interesting that RTX-VII did not bind to Nav1.3 DII. Therefore, the role of this domain in the toxin-induced INaP remains unclear. Previous studies showed that the inter-domain interactions of Navs is necessary for channel gating50,51. We proposed in this study that the DII and DIV of Nav1.3 might cooperate to trigger late brief opening and burst of opening to generate INaP and RTX-VII should facilitate/amplify this cooperation to induce large INaP in Nav1.3. The subtle amino acid sequence differences of the domain II between Nav1.3 and Nav1.5 greatly affect this cooperation, namely the DII of Nav1.3 can cooperate well with its own DIV, which is not the case for the DII of Nav1.5 with DIV of Nav1.3. The roles of Nav1.3 DI and DIII of in the toxin-induced INaP generation were unclear. The fact that the replacement of the DI or DIII of Nav1.3 with that of Nav1.5 did not affect toxin efficacy could not exclude the possibility that both domains might involve in the INaP generation, because high sequence similarity of DI and DIII between Nav1.3 and Nav1.5 is observed and probably the inter-domain interactions might not be interfered although these two domains were replaced.

RTX-VII induced large INaP in Nav1.3 at the end of a 50-ms or a 300-ms depolarization, which differs from some scorpion toxins and sea anemone toxins that slow the inactivation of Navs but the resultant current decay rapidly in 50 ms (Lqh2 as a representative52). What is the difference derived from? Theoretically, Lqh2 trapping the DIV-VSD of Navs in the closed state should have induced large INaP, but the fact is not. How are the toxin-bound channels inactivated? Slow inactivation may not be the underling mechanism. This is because that slow inactivation is rarely observed in a 50 ms depolarization (such a short depolarization is not sufficient to trigger this gating process). The repriming kinetics of the toxin-bound channels is the same as or even faster than that of the toxin-free channels37, which is also inconsistent with the fact that the recovery of Navs from slow inactivation is slow53. Based on the unique role of DIV in fast inactivation, a model was proposed to clarify these two problems. Macroscopically, in this model, a depolarization would drive and maintain the first three domains of Navs in an activated state; Lqh2 and RTX-VII could trap the DIV-VSD of Navs in the closed52 and partially activated state, respectively. For Lqh2, such trapping is not very stable, as the depolarization prolongs, the toxin-bound DIV-VSD would be gradually activated, triggering channel inactivation. However, for RTX-VII, the DII of Nav1.3 might allosterically slow/inhibit this process, which therefore makes RTX-VII stably trap the DIV-VSD of Nav1.3 in the partially activated state and then the channels would maintain a persistent opening state. The understanding of this process in the single channel level could be as follows: the inactivation ball of a Nav has a “on state” (blocking the pore) and an “off state” (free in cytosol) which are tightly coupled to the activated and resting state of DIV-VSD, respectively8. Normally, DIV-VSD is immobilized in an outward conformation by activation54,55. The toxin-bound DIV-VSD could be activated by strong depolarization but not be stably immobilized as toxins tend to “drag” the DIV-VSD to its resting state (partially activated state for RTX-VII). Thus, when the toxin-bound DIV-VSD is activated, the inactivation ball is in the “on state” and the pore is occluded; when the toxin-bound DIV-VSD is in the resting state (partially activated state for RTX-VII), the channel just opens. The inactivation ball switches between the “on state” and the “off state” quickly and such inactivation ball movement should trigger the burst opening of the channel in single channel recording. For Lqh2, as the depolarization prolonged, the DIV-VSD of most channels would be stably immobilized and these channels were consequently trapped stably in the inactivated state. On the other hand, for RTX-VII, toxin binding to the DIV-VSD of Nav1.3 should allosterically affect the conformation of DII-VSD, which would in turn interfere with the time-dependent immobilization of DIV-VSD. We proposed that such gating model underlies the generation of large INaP in Nav1.3 by RTX-VII.

Methods

Venom and toxin purification

Spider Macrothele raveni were collected in GuangXi province, China. The spider has a body length of 3–5 cm and the venom was collected by an electric stimulation method as described in another work of our laboratory34. The collected crude venom was lyophilized and preserved at −80°C before use. The crude venom was dissolved in ddH2O to a final concentration of 5 mg/ml and subjected to the first round of RP-HPLC purification (acetonitrile gradient: 1%–60%, at an increasing rate of 1% per minute). The fraction containing RTX-VII was then collected, lyophilized and subjected to the second round of RP-HPLC with a slower increasing acetonitrile gradient (acetonitrile at an increasing rate of 0.5% per minute) to obtain the purified toxin.

Toxin sequencing and cDNA of RTX-VII

Partial amino acid sequence of RTX-VII was determined by Edman degradation on an Applied Biosystems/PerkinElmer Life Science Procise 491-A protein sequencer. The cDNA of this toxin was obtained by blasting Edman degradation determined amino acid sequence of RTX-VII against the local cDNA library database of the spider Macrothele raveni (unpublished data).

Constructs and transfection

All Nav clones and beta subunit clones were kindly gift from Dr Theodore R.Cummins (Department of pharmacology and Toxicology, Stark Neurosciences Research Institute, Indiana University School of Medicine, USA). cDNA genes encoding rat Nav1.3 and rat Nav1.4 were subcloned into the vectors pcDNA3.1 and pRGB440,56, respectively; the cDNA genes encoding human Nav1.5 and human Nav1.7 were subcloned into the vectors pcDNA3.1 and pcDNA3.1-mod57, respectively. Auxiliary β1 and β2 subunits both were cloned from human and inserted into an internal ribosome entry site vector58. All site mutations of Nav1.3 were constructed by using the QuikChange II XL Site-directed Mutagenesis kit (Agilent Technologies) according to the manufacture's instruction. The cytosolic boundaries of two adjacent transmembrane segments and two adjacent domains of Nav1.3 or Nav1.5 were determined by proteins' topological information deposited in NCBI protein database (for Nav1.3, the website is http://www.ncbi.nlm.nih.gov/protein/NP_037251.1 and for Nav1.5,the website link is http://www.ncbi.nlm.nih.gov/protein/NP_932173.1). The protein sequence location of each voltage sensor (VSD)/pore domain (PD) of all four domains of Nav1.3 and Nav1.5 are as listed in Supplementary Table S4. A homologous recombination strategy was employed to generate the chimeric channels using the In-FusionHD Cloning kit (Clontech Laboratories) or CloneEZ PCR Cloning kit (Genscript). For example, for the construction of Nav1.3/1.5 DI-VSD chimera, the DI-VSD (voltage sensor of domain I) of Nav1.5 was amplified by PCR using a pair of primers with their 5′ end extended by a 15 bp long joint which is homologous or reverse compliment to the upstream or downstream flanking sequence of DI-VSD of Nav1.3. A pair of oppositely directed primers was used to linearize the whole Nav1.3 cloned plasmid with the DI-VSD of Nav1.3 deleted. The PCR amplified segment and the linearized plasmid were subjected to 1% agarose gel electrophoresis, respectively. The corresponding bands were recycled using a DNA gel extraction kit (Sangon biotech) and ligated using the In-FusionHD Cloning kit (Clontech Laboratories) or CloneEZ PCR Cloning kit (Genscript). Before being transformed to E.coli Top10 competent cell, the ligated product was subjected to FastDigest DpnI (Thermo Scientific) treatment at 37°C for 1 hour to remove the template plasmid. The transformants were verified by colony-PCR using a pair of gene specific primer for each inserted segment and then sequencing (Genscript). The primers used for vector linearization and amplification of Nav domains were listed in Supplementary Table S2 and Table S3. HEK293T cells (ATCC) were grown under the standard cell culture conditions (5% CO2 and 37°C) in Dulbecco's Modified Eagle Medium (DMEM, Life technologies) supplemented with 10% fetal bovine serum. These Nav constructs were co-transfected with plasmid containing β1 subunit and PEGFP-N1 to HEK293T cells using Lipofectamine 2000 (Life Technology) according to the manufacture's instruction. For wt-Nav1.3, Nav1.3 mutants and Nav1.3 derived chimeric channels, 3 μg Nav plasmid, 1 μg plasmid containing β1 subunit and 0.5 μg PEGFP-N1 plasmid were co-transfected. For wt-Nav1.5 and Nav1.5 derived chimeric channels, 1 μg Nav plasmid, 0.3 μg plasmid containing β1 subunit and 0.5 μg PEGFP-N1 plasmid were co-transfected. For ramp test, Nav1.3 was co-transfected with plasmid containing β1 subunit and plasmid containing β2 subunit33. Cells were 80%–90% confluent before transfection and cells were seeded on a poly-lysine coated Microscope Cover Glass (Fisher scientific) 4–6 hours after transfection. 24 hours after seeding, cells were ready for patch-clamp analysis.

Primary culture of DRG and hippocampal neurons and toxicity test of animals

Animals (Sprague-Dawley rats and Kunming mice) were used according to the guidelines of the National Institutes of Health for care and use of laboratory Animals. The experiments were approved by the Animal Care and Use Committee of the College of Medicine, Hunan Normal University. Acutely dissociated dorsal root ganglion (DRG) cells were prepared from 4 weeks old Sprague-Dawley rats and maintained in short-term primary culture using the method described by Hu, H.Z and Li, Z.W59. The dissociated cells were suspended in DMEM supplemented with 10% fetal bovine serum, 50 IU/ml penicillin and 50 μg/ml streptomycin. Cells were seeded on poly-L-lysine-coated Microscope Cover Glass placed in a cell culture dish (35 × 10 mm, corning) and incubated at 37°C in an atmosphere of 5% CO2. Cells cultured for 3–24 h were used in the patch experiments. Experiments were conducted at room temperature (20–25°C). For primary culture of hippocampal neurons, hippocampal tissues of neonatal rats were dissected and treated with 0.25% trypsin in Ca2+-Mg2+-free Hank's Buffered Salt solution at 37°C for 15 min and then were dissociated by trituration with glass Pasteur pipette and seeded on poly-L-lysine-coated Microscope Cover Glass placed in a cell culture dish (35 × 10 mm, corning). Approximate 3.5*104 cells in DMEM containing 10% fetal bovine serum were plated in each dish. The culture medium were replaced with serum-free Neurobasal medium (Life technologies) supplemented by 2% B27 (Life technologies) on the second day after plating, 500 μM glutamine was added to reduce the growth of glial cells. The hippocampal neurons were maintained in a CO2 incubator at 37°C, one-half volume of the culture medium was replaced with fresh medium every other day. The neurons were used for patch-clamp analysis after they were maintained in culture for 14–17 days. In order to test the neurotoxicity of RTX-VII, ten mice of either sex with an average weight of 20 g were randomly divided to two groups, animals in the control group were intracerebroventricularlly injected with 20 μL saline and animals in the experimental group were injected with 20 μL saline containing 20 ng toxin.

Electrophysiology

Cell current recording was made with the whole-cell patch-clamp technique using an EPC 10 USB Patch Clamp Amplifier (HEKA Elektronik). Cells transfected with wt/mutant/chimeric Nav channels and DRG/hippocampal neurons seeding in a glass coverslip were placed in a perfusion chamber in which rapid exchange of solutions around cells could be performed. The recording pipettes were made from glass capillary (thickness = 0.225 mm) using a PC-10 puller (NARISHIGE).The pipet resistance was controlled at 1.5–2.0 MΩ by adjusting the pulling temperature. The standard pipet solution contained (in mM): 140 CsCl, 10 NaCl, 1 EGTA, 2 Mg-ATP and 20 HEPES (pH 7.4). Bath solution contained (in mM): 140 NaCl, 2 CaCl2, 1 MgCl2, 5 KCl, 20 HEPES (pH 7.4) and 10 glucose. All experiments were conducted at the room temperature (20–25°C). All chemicals were the products of SigmaAldrich and dissolved in water. Data were acquired by PatchMaster software (HEKA Elektronik). Data were analyzed by softwares Igo Pro 6.10A, Excel 2010, Sigmaplot 10.0 and OriginPro 8. Voltage errors were minimized by using 80% series resistance compensation, the speed value of Rs compensation was set to be 10 μs(fast compensation).The capacitance artifact were canceled using the computer-controlled circuitry of the patch clamp amplifier. The pipet capacitance was minimized by filling the pipet with small volume of pipet solution and the pipet capacitance was controlled to be <10 pF for effective automatic compensation by EPC-10 amplifier. The pipet capacitance and the cell capacitance was sequentially compensated after the seal and the whole-cell configuration was established, respectively(pipet capacitance and cell capacitance was compensated by automatic fast and slow capacitance compensation, respectively).Stock solution of RTX-VII (1 mM in sterile ddH2O) was diluted with fresh bath solution to a concentration of 10 folds of the interested concentration, 30 μL of the concentrated toxin was diluted into the recording chamber (containing 270 μL bath solution) far from the recording pipet (the recording cell) and was mixed by repeatedly pipetting to achieve the specified final concentration60. The dose-response curves of toxin on wt/mutant/chimeric channel were fitted to a Hill equation to estimate the potency of toxin (EC50). The G-V curve before and after toxin treatment and the steady state inactivation (SSI) curve before toxin treatment were fitted using a boltzmann equation: y = 1/(1 + exp[(V1/2 − V)/K]) in which V1/2, V and K represented midpoint voltage of kinetics, test potential and slope factor, respectively. The SSI curve after toxin treatment was fitted with a modified Boltzmann equation: (Y − Ymin)/(Ymax − Ymin) = 1/(1 + exp[(V1/2 − V)/K)]), Ymax and Ymin represent the maximum and minimum responses. The time course curve for the INaP enhancement in response to the toxin application was best fitted by a single exponential rising equation (y = y0 + a(1 − e−x/τ)) and the time course for recovery of INaP upon bath solution washing was best fitted by a single exponential decay equation(y = y0 + ae−x/τ), here τ represent the time constant for toxin binding to and washing off from channels respectively. In measuring the spontaneous AP firing of neonatal rat hippocampal neuron using current-clamp, the pipette solution contains (in mM): 140 KCl, 5 MgCl2, 5 EGTA, 2.5 CaCl2, 4 ATP, 0.3 GTP and 10 Hepes, pH 7.3 (adjusted with KOH). The bath solution contains (in mM): 140 NaCl, 1 MgCl2, 5 KCl, 2 CaCl2, 10 HEPES and 10 glucose, pH 7.3 (adjusted with NaOH). During the recording, no current was injected to neurons.

Data analysis

Data were presented as Mean ± SD. n is presented as the number of the separate experimental cells. Dose response curves were fitted using the following Hill logistic equation: y = fmax − (fmax − fmin)/(1+([Tx]/EC50)n), where fmax and fmin represent the maximum and minimum response of channel to toxin, [Tx] represent the toxin concentration, n is an empirical Hill coefficient. The Hill coefficient was set to 1 except where indicated otherwise. This is reasonable based on our mutagenesis analysis, which indicated a single high affinity binding site in Nav1.3 for RTX-VII. Statistical significance was assessed with Microsoft excel 2010 using One-Way ANOVA. Statistical significance was accepted at P values less than 0.05.