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

Abstract

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 Na(v)1.8, a tetrodotoxin (TTX)-resistant sodium channel, in rat small-diameter DRG neurons employing whole-cell patch clamp recordings. NK-1 agonist [Sar(9), Met(O2)(11)]-substance P (Sar-SP) significantly enhanced the Na(v)1.8 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 PKCepsilon, 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 Na(v)1.8 sodium current via PKCepsilon-dependent signaling pathway, probably participating in the generation of inflammatory hyperalgesia.

Figure 1

Recording of Na_v1.8 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_v1.8 currents. B: I-V curve of Na_v1.8 currents shown in (A). C: representative traces of Na_v1.8 current elicited by a single pulse of -10 mV which was used in most of the recordings. D: peak amplitudes of Na_v1.8 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).

Figure 2

Effect of NK-1 agonist Sar-SP on Na_v1.8 currents. A: time course of the potentiation effect of Sar-SP. The maximal increase in the peak amplitude was at 3 min after Sar-SP perfusion, and reduced slowly to control level thereafter. B: typical traces illustrating the Na_v1.8 current recorded in a neuron pre- (dashed line, control) and post- (solid line, Sar-SP) perfusion of 1 μM Sar-SP. C: histogram showing the effect of 1 μM and 10 μM Sar-SP. The normalized peak current was enhanced to 116.2 ± 2.9% and 117.1 ± 1.4% 3 min after perfusion of 1 μM and 10 μM Sar-SP, respectively (***p < 0.001, versus control, Kruskal-Wallis one-way ANOVA, n = 16 for control, 13 for 1 μM, and 6 for 10 μM). D: NK-1 antagonist Win51708 (5 μM) completely blocked the effect of Sar-SP in all 15 neurons tested (p > 0.05, t-test). E: The rate of the Sar-SP-responsive cells was increased after CFA-treatment. F: The effect of Sar-SP was also increased after peripheral CFA-treatment (* p < 0.05, t-test, n = 8 for saline and 14 for CFA-treated). G and H: Sar-SP shifted the activation (G) and steady-state inactivation (H) curve in a hyperpolarizing direction.

Figure 3

Involvement of PKC, but not PKA in Sar-SP-induced potentiation of Na_v1.8 currents. Incubation with PKC inhibitor BIM (1 μM) for 30 min before Sar-SP perfusion completely blocked the potentiation effect of Sar-SP in all 24 neurons tested (A and B, p > 0.05, Mann-Whitney rank sum test). After incubation with PKA inhibitor H89, Sar-SP (1 μM) still fully enhanced Na_v1.8 currents in 9 of 24 DRG neurons (C and D, ***p < 0.001, Mann-Whitney rank sum test).

Figure 4

PMA mimic the effect of Sar-SP. A and B:representative traces (A) and histogram (B) showing the effect of PMA on Na_v1.8 currents. 300 nM PMA induced a similar potentiation to 1 μM Sar-SP. Perfusion with Sar-SP failed to further enhance the currents after PMA-induced peak potentiation (***p < 0.001, versus control, Kruskal-Wallis one-way ANOVA, n = 16 for control, 10 for PMA and 10 for Sar-SP). PMA also shifted the activation and steady-state inactivation curve of Na_v1.8 in a hyperpolarized direction (C and D).

Figure 5

PKCε was the main PKC subtype mediating the effect of Sar-SP. A and B: representative traces (A) and histogram (B) showing the effect of PKCε inhibitor εV1-2 on Sar-SP-induced potentiation. Intracellular application of εV1-2 (200 μM) completely abolished the potentiation effect of Sar-SP. The negative control (control peptide) of εV1-2 failed to block Sar-SP-induced potentiation (***p < 0.001, versus normal control, one-way ANOVA, n = 16 for normal control, 11 for εV1-2 and 8 for negative control).

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).