PKCε-dependent potentiation of TTX-resistant Nav1.8 current by neurokinin-1 receptor activation in rat dorsal root ganglion neurons

Background Substance P (SP), which mainly exists in a subtype of small-diameter dorsal root ganglion (DRG) neurons, is an important signal molecule in pain processing in the spinal cord. Our previous results have proved the expression of SP receptor neurokinin-1 (NK-1) on DRG neurons and its interaction with transient receptor potential vanilloid 1 (TRPV1) receptor. Results In this study we investigated the effect of NK-1 receptor agonist on Nav1.8, a tetrodotoxin (TTX)-resistant sodium channel, in rat small-diameter DRG neurons employing whole-cell patch clamp recordings. NK-1 agonist [Sar9, Met(O2)11]-substance P (Sar-SP) significantly enhanced the Nav1.8 currents in a subgroup of small-diameter DRG neurons under both the normal and inflammatory situation, and the enhancement was blocked by NK-1 antagonist Win51708 and protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM), but not the protein kinase A (PKA) inhibitor H89. In particular, the inhibitor of PKCε, a PKC isoform, completely blocked this effect. Under current clamp model, Sar-SP reduced the amount of current required to evoke action potentials and increased the firing rate in a subgroup of DRG neurons. Conclusion These data suggest that activation of NK-1 receptor potentiates Nav1.8 sodium current via PKCε-dependent signaling pathway, probably participating in the generation of inflammatory hyperalgesia.


Background
Substance P (SP), a member of tachykinin family, is a well-known pain-related neuropeptide in the spinal cord. It is released by unmyelinated primary afferent fiber terminals of small-diameter dorsal root ganglion (DRG) neurons and participates in the spinal transmission of nociceptive signals [1][2][3]. It is well documented that the SP receptor neurokinin-1 (NK-1) is densely distributed in the superficial dorsal horn and involved in the development of chronic pain and central sensitization after intense noxious stimulation and tissue/nerve injury [4][5][6][7].
In addition to the expression of the NK-1 on the postsynaptic neurons of superficial spinal dorsal horn, increasing evidence strongly suggested the presynaptic expression of NK-1 in DRG neurons. The immunohistochemical evidence revealed that the NK-1 was expressed by the unmyelinated axons of the glabrous skin [8], and the DRG neuron soma in rats [9]. By means of intracellular and whole-cell patch clamp recordings, SP was shown to be able to induce the depolarization of DRG or trigeminal ganglion neurons in the different species [10][11][12][13] and potentiated the TRPV1 currents [9]. However, the function of DRG-expressed NK-1 receptor needs to be further understood.
The Na v 1.8, which is a TTX-resistant sodium channel and mainly expressed in small-diameter DRG neurons [14,15], is a major contributor to the upstroke of action potential in these neurons [16]. In the Na v 1.8-null mice or Na v 1.8 knockdown mice by antisense oligodeoxynucleotides, both the physiological and pathological pain was alleviated [17][18][19][20]. Accumulative evidence showed that the Na v 1.8 current was regulated by various inflammatory mediators, such as prostaglandin E 2 (PGE 2 ), serotonin, NGF etc. through a PKA or PKC signaling pathway [21][22][23][24].
In the present study, we investigated the effects of the NK-1 agonist on dynamics of Na v 1.8 currents in isolated small-diameter DRG neurons using whole-cell patch clamp recording. Also, the role of PKC signal pathway in the cross-talk between NK-1 and Na v 1.8 was examined.

Recording of Na v 1.8 currents in DRG neurons
With existence of TTX (500 nM) in external solution, TTXresistant sodium currents were recorded in most (166 out of 205) of the small-diameter DRG neurons (<25 μm). The membrane potential was hold to -60 mV. Under this recording condition, TTX-resistant sodium currents were mainly mediated by Na v 1.8 channels due to inactivation of Na v 1.9 [25,26]. The family of Na v 1.8 sodium currents was generated with a voltage-clamp protocol (depolarizing steps from -55 mV to 40 mV, 50 ms, 5 mV increments, Figure 1A). In accordance with the current-voltage relationship ( Figure 1B), -10 mV was chosen to elicit Na v 1.8 currents in most of the recordings ( Figure 1C). As reported by Saab et al. [27], fluoride-based pipette solution also caused slow stabilization of the amplitude of Na v 1.8 current after rupture of cell membrane in our experiments. We measured the peak amplitude of the Na v 1.8 current at 5 min, 10 min and 15 min after whole-cell mode was performed. As shown in Figure 1D, the peak amplitude was relatively stable from 5 min to 15 min (n = 16). All of our subsequent recordings were performed in this time course.

Increase in peak amplitude of Na v 1.8 current by Sar-SP
Following the perfusion with Sar-SP (1 μM, 1 min), a selective NK-1 agonist, Na v 1.8 currents were increased in 13 out of 30 DRG neurons tested (Figure 2A, B and 2C). The maximal enhancement of the peak amplitude occurred at 3 min after Sar-SP perfusion, and reduced slowly to control level thereafter ( Figure 2A). As shown in Figure 2C, normalized currents were increased significantly by Sar-SP (116.2 ± 2.9%, n = 13) compared with the control (99.2 ± 0.5%, n = 16, p < 0.001). Higher concentration of Sar-SP (10 μM) failed to induce more powerful action (117.1 ± 1.4%, n = 6, p < 0.001, Figure 2C), suggesting a "ceiling effect" at 1 μM. The Sar-SP-induced potentiation was blocked by co-incubation of Win51708, a selective NK-1 antagonist (5 μM, 98.5 ± 0.6%, n = 15, Figure 2D). To exclude the influence of fluoride, we also test the effect of Sar-SP by using chloride-based pipette solution. Cesium fluoride was changed to cesium chloride. The effect of Sar-SP wasn't changed significantly (data not shown).
The effect of Sar-SP on Na v 1.8 current was also examined under peripheral inflammation condition. Complete Freund's adjuvant (CFA, 100 μl) was bilaterally injected into rat hind paws. DRG neurons was tested three days after CFA treatment, Na v 1.8 currents were enhanced by Sar-SP (1 μM) in 60.9% of DRG neurons (n = 23). In the saline treated control rats, 38.1% of DRG neurons (n = 21) exhibited potentiation of Na v 1.8 current ( Figure 2E). In rats with CFA treatment, the peak Na v 1.8 current in DRG neurons was enhanced to 123.2 ± 1.8% (n = 14) following application of Sar-SP, whereas enhanced to 117.4 ± 1.5% (n = 8) in saline treated control rats ( Figure 2F, p < 0.05). Given inflammation-induced increase in expression of NK-1 [9], it is suggested that such modulation of NK-1 may be more beneficial for controlling inflammatory chronic pain.

Sar-SP shifted the activation and steady-state inactivation curves of Na v 1.8 in a hyperpolarizing direction
As described above, a voltage-clamp protocol consisted of 50 ms depolarizing steps from -55 mV to 40 mV (5 mV increments) was used to determine the activation of Na v 1.8 channels. Sar-SP caused a left shift of the activation curve ( Figure 2G). Half-maximal activation potential (V 1/ 2activation ) was shifted to -20.03 ± 0.23 mV from that of control condition (-14.24 ± 0.25, n = 6) after Sar-SP perfusion. The k value was not changed by Sar-SP (k control = 4.89 ± 0.27, k Sar-SP = 4.77 ± 0.45). Steady-state inactivation was determined at a series of membrane potentials from -60 mV to 15 mV (5 mV increments) for 500 ms and a following test potential of 20 mV. Similar to the activation curve, the steady-state inactivation curve was also shifted in a hyperpolarized direction after Sar-SP perfusion (Figure 2H). The V 1/2 of voltage dependence of steady-state inactivation was -19.31 ± 0.23 mV (n = 6) using vehicle alone, and -25.55 ± 0.14 mV after Sar-SP treatment (n = 6). The k value was also not changed by Sar-SP (k control = 5.39 ± 0.37, k Sar-SP = 5.09 ± 0.32).

Sar-SP-induced potentiation of Na v 1.8 via PKC, but not PKA
Activation of NK-1, a G-protein coupled receptor, triggers several intracellular signal pathways, such as PKA and PKC pathway [28]. Given modulation of Na v 1.8 by PKC [23,24,29], here we used bisindolylmaleimide (BIM), a PKC inhibitor, to examine its effect on the Sar-SP-induced potentiation of Na v 1.8 currents. In all DRG neurons (n = 24) incubated with BIM (1 μM) for 30 min before Sar-SP perfusion, Sar-SP failed to potentiate Na v 1.8 currents, suggesting a PKC-dependent mechanism in the interaction of NK-1 and Na v 1.8 ( Figure 3A and 3B).
Na v 1.8 currents were also reported to be regulated by PKA [22,23,30]. While the cells were incubated with the PKA inhibitor H89 (1 μM) for 30 min, Sar-SP (1 μM) still fully enhanced Na v 1.8 currents in 9 of 24 DRG neurons ( Figure  3C and 3D, control = 99.2 ± 0.5%, Sar-SP = 115.6 ± 2.1%, Recording of Na v 1.8 currents in rat DRG neurons Figure 1 Recording of Na v 1.8 currents in rat DRG neurons. A: representative I-V curve family of currents recorded in the presence of 500 nM TTX is shown, using a protocol (inset) where cells were depolarized to a variety of potentials (-55 to +40 mV) from a holding potential of -60 mV to elicit Na v 1.8 currents. B: I-V curve of Na v 1.8 currents shown in (A). C: representative traces of Na v 1.8 current elicited by a single pulse of -10 mV which was used in most of the recordings. D: peak amplitudes of Na v 1.8 currents elicited by -10 mV pulse at 5 min, 10 min and 15 min after whole-cell mode was performed. The currents were stable during the recording time in all the cells (n = 16).
p < 0.001), suggesting that the PKA pathway did not participate in the interaction of NK-1 and Na v 1.8.

PKCε: a pivotal factor for potentiation of Na v 1.8 by NK-1 activation
As reported, there are five isoforms of PKC expressed in DRG neurons [31]. Among these isoforms, PKCε is highly expressed in small-diameter DRG neurons and involved in NK-1 activation-induced potentiation of TRPV1 and the development of hyperalgesia [9,[31][32][33]. Therefore, to explore the effect of PKCε on Sar-SP-induced potentiation of Na v 1.8, εV1-2 (200 μM), a specific PKCε inhibitor, was delivered intracellularly via recording electrodes. The potentiation of Na v 1.8 currents was completely blocked Effect of NK-1 agonist Sar-SP on Na v 1.8 currents by εV1-2 (n = 15), but not by its negative control (control peptide) (n = 8, Figure 5A and 5B).

Effect of Sar-SP on excitability of DRG neuron
Na v 1.8 is the main contributor to the upstroke of action potentials in small-diameter DRG neurons [16]. Therefore, modulation of this channel by Sar-SP should influence excitability of the DRG neuron. We detected the effect of Sar-SP on the threshold for evoking action potential in DRG neurons. To evoke action potentials, 5 ms step depolarizing current pulses were applied to neurons before and after exposure to Sar-SP ( Figure 6A and 6B). In 6 of 17 neurons tested, Sar-SP significantly decreased the injected current threshold to evoke action potentials. Under the control condition, the current threshold was 116.7 ± 7.6 pA. After exposure to Sar-SP, this threshold was reduced to 80.0 ± 5.2 pA ( Figure 6C, n = 6, p < 0.01).

Discussion
The role of substance P (SP) and its NK-1 receptor in pain processing was widely investigated in the spinal cord. However, most of the prevailing studies focused on the postsynaptic NK-1 receptors in the spinal superficial dorsal horn neurons. Whether NK-1 receptors are also Involvement of PKC, but not PKA in Sar-SP-induced potentiation of Na v 1.8 currents expressed presynaptically in primary sensory neurons is still obscure. A growing body of evidence showed that SP could activate DRG neurons through NK-1 receptor in primary sensory neurons [10][11][12][13]34,35], in despite of a contradictory report [36]. Our recent study provided new evidence for the expression of NK-1 receptor protein and interaction with TRPV1, a crucial pain signal molecule, in DRG neurons [9].
In addition to TRPV1, another important ion channel, TTX-resistant sodium channel, is also primarily expressed in nociceptors. Between the two distinct TTX-resistant sodium channel isoforms Na v 1.8 and Na v 1.9, Na v 1.8 likely mediates the majority of the TTX-resistant currents and plays an important role in pain processing. Na v 1.8null mice displayed a pronounced increase in threshold to noxious mechanical stimuli and a slight decrease in nociceptive thermoreception as well as delayed development of inflammatory hyperalgesia [17]. Likewise, knockingdown of Na v 1.8 mRNA with antisense oligodeoxynucleotides was effective in alleviating both the inflammatory and neuropathic pain [18][19][20]37,38]. Also, muO-conotoxin MrVIB, a selective blocker of Na v 1.8, reduced allodynia and hyperalgesia in neuropathic and chronic inflammatory pain models [39,40]. The present study for the first time revealed that NK-1 activation potentiated Na v 1.8 currents and shifted both the activation and steady-state inactivation curves of this channel in a hyper- polarizing direction. This change in voltage sensitivity of Na v 1.8 may decrease the activate threshold and increase the likelihood of action potential firing, and then probably cause a hyperexcitability of the neurons. As shown in Figure 6, the enhancement of excitability was observed in our experiments. Although the involvement of Sar-SPinduced modulation on other ion channels still need to be further investigated, it is assumed that modulation on Na v 1.8 at least partly contributes to this enhancement of excitability. Similar results were also obtained from studies on modulation of Na v 1.8 by another peripheral painrelated neuropeptide calcitonin gene related peptide (CGRP) [41] and proinflammatory factors such as 5hydroxytryptamine and prostaglandin E 2 [22,42,43]. It is conceivable that the modulation of Na v 1.8 by NK-1 activation may contribute to peripheral sensitization of pain pathway.

PMA mimic the effect of Sar-SP
NK-1 receptor is a G-protein coupled receptor [28]. The activation of NK-1 receptors generates various second messengers, which, in turn, trigger a wide range of effector mechanisms underlying regulating cellular excitability and functions [44][45][46][47]. In agreement with our previous finding that the modulation of TRPV1 by NK-1 receptor was mediated by activation of PLC and downstream PKC pathway [9], the present results also proved the involvement of PKC in the interaction between NK-1 and Na v 1.8. Figure 3 and Figure 4, PKC inhibitor BIM completely blocked Sar-SP-induced potentiation of Na v 1.8 currents, whereas PKC activator PMA could mimic the effects of Sar-SP on Na v 1.8 currents. These results suggest that NK-1 modulates Na v 1.8 in a PKC-dependent pathway. There are many other papers confirmed the enhancement of Na v 1.8 by PKC pathway [24,48,49]. However, the inconsistent results have been reported. Gold et al. reported that PKC activation also caused an increase in the amplitude of the TTX-resistant current in rat DRG neurons. But this increase was not associated with a shift in the activation curve [23]. Vijayaragavan and colleagues reported that in Xenopus oocytes expression system, PKC activator PMA caused a decrease of Na v 1.8 current and a right shift of the activation curve [29]. The reason for the difference is still unclear.

As shown in
Furthermore, we observed that PKCε inhibitor εV1-2 completely blocked Sar-SP-induced potentiation of Na v 1.8 currents, suggesting that PKCε was the main mediator of NK-1 potentiation, in consistence with the modulation of TRPV1 by NK-1 [9].
In addition to the PKC pathway, several reports showed that PKA was also involved in the increase of TTX-resistant currents by proinflammatory agents (5-HT, PGE 2 ) [22,23,42]. However, PKA inhibitor H89 failed to prevent PKCε was the main PKC subtype mediating the effect of Sar-SP Sar-SP-induced potentiation of Na v 1.8 in the present study. These suggested the diverse mechanisms underlying modulation of Na v 1.8 by the different proinflammatory agents. Therefore, the modulatory action of NK-1 may be predominately mediated by PKC, particularly by PKCε, but not PKA.
It is well documented an increase in expression of Na v 1.8 in DRG neurons in several inflammatory pain models [50-53]. Our previous results have revealed that both the NK-1 expression and phosphorylation of PKCε are up-regulated in DRG after CFA-induced inflammation [9,33]. Therefore, we assume that the modulation of Na v 1.8 by NK-1 via PKCε is likely to be stronger after peripheral inflammation. In support of this view, the present study showed that not only the effect of NK-1 activation on Na v 1.8 currents was significantly potentiated, but also the rate of Sar-SP-responsive neurons following CFA treatment. It is conceivable that the modulation of Na v 1.8 by NK-1 may amplify peripheral nociceptive inputs and in turn strengthen activation of the pain-sensitive neurons in the spinal cord, contributing to inflammatory pain.

Conclusion
Substance P (SP) receptor NK-1 and TTX-resistant sodium channel Na v 1.8 expressed on nociceptors are two important molecules for pain processing. The present study for the first time investigated their interaction in rat DRG neurons. The results showed that activation of NK-1 receptor potentiates Na v 1.8 sodium current via PKCε-dependent signaling pathway, probably participating in the generation of inflammatory hyperalgesia.
Effect of 1 μM Sar-SP on action potential threshold and firing rate in DRG neurons Figure 6 Effect of 1 μM Sar-SP on action potential threshold and firing rate in DRG neurons. Sar-SP reduced the amount of current required to evoke action potential and increased the firing rate in DRG neurons. Experiments were performed using current clamp model. A and B: depolarizing current pulse required to evoke an action potential in a DRG neuron, before (A) and after (B) application of Sar-SP (a = 130 pA, b = 140 pA, c = 80 pA, d = 90 pA). C: effect of Sar-SP on the threshold for action potential generation by depolarizing current pulse (**p < 0.01, paired t-test, n = 6). D and E: firing response of a DRG neuron to a 500 pA depolarizing current pulse (500 ms), before (D) and after (E) application of Sar-SP. F: effect of Sar-SP on firing rate in DRG neurons (***p < 0.001, paired t-test, n = 6).

Animals
Male adult (100-150 g) Sprague-Dawley rats (obtained from the Experimental Animal Center, Shanghai Medical College of Fudan University, China) were used in our experiments. Rats were on a 12 h light/dark cycle with a room temperature of 22 ± 1°C and received food and water ad libitum. All experimental procedures were approved by the Shanghai Animal Care and Use Committee and followed the policies issued by the International Association for the Study of Pain on the use of laboratory animals. All efforts were made to minimize animal suffering and reduce the numbers of animals used.

Cell preparation
Culture of DRG neurons was established as described previously [33]. Briefly, DRGs from L 4 -L 6 lumbar segments were dissected and incubated at 36.8°C for 25 min in DMEM containing 3 mg/ml collagenase (type IA, Sigma, St. Louis, MO) and, 1 mg/ml trypsin (type I, Sigma). The ganglias were then gently triturated using fine fired-polished Pasteur pipettes. The dissociated DRG neurons were plated onto coverslips (10 mm diameter) in the 3.5 cm culture dishes and incubated with Standard external solution containing (in mM) 150 NaCl, 5 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 HEPES, and 10 glucose, adjusted to pH 7.4 with NaOH.

Drugs
All the drugs were purchased from Sigma (St. Louis, MO, USA), except that the PKCε inhibitor εV1-2 and its negative control were from Biomol (Plymouth Meeting, PA). All the drugs are prepared on the day of the experiment from stocks kept at -20°C at a concentration at least 1000fold the working concentration. [Sar 9 , Met(O 2 ) 11 ]-substance P (Sar-SP) and PMA were applied close to the cells through a ALA-VM8 perfusion system (ALA Scientific Instruments, Westbury, NY). Inhibitors were applied (where appropriate) to the chamber for 30 min before the perfusion of Sar-SP and PMA and existed during the whole recording course.

Data analysis
Peak sodium current values were converted to conductance values using the equation: G = I/(V m -E Na ), where G is the conductance, I is the peak current amplitude, V m is the membrane potential, and E Na is the equilibrium potential value for Na + . The Boltzmann equation used to describe the voltage dependence of activation was of the form: G/G max = 1/(1 + exp [(V 1/2 -V m )/k]), where G max is the peak conductance, V 1/2 is the potential at half maximal activation, and k is the slope factor. Voltage dependence of steady-state inactivation was described by the Boltzmann function: I/I max = 1/(1 + exp [(V-V 1/2 )/k]), where I max is the maximal peak current, V is the prepulse membrane potential, V 1/2 is the potential at half maximal activation, and k is the slope factor.
Data are expressed as means ± standard error of the mean (SEM). Statistical analysis was performed using SigmaStat software (Systat Software, Chicago, IL). Student's t-test or Mann-Whitney analysis was used to assess differences between means from two groups. One-way ANOVA or Kruskal-Wallis one-way ANOVA was used to assess difference among more groups. P < 0.05 was considered significant. Curves were plotted and fitted using Origin software (OriginLab Corporation, Northampton, USA).