The role of slow and persistent TTX-resistant sodium currents in acute tumor necrosis factor-α-mediated increase in nociceptors excitability

Abstract

Tetrodotoxin-resistant (TTX-r) sodium channels are key players in determining the input-output properties of peripheral nociceptive neurons. Changes in gating kinetics or in expression levels of these channels by proinflammatory mediators are likely to cause the hyperexcitability of nociceptive neurons and pain hypersensitivity observed during inflammation. Proinflammatory mediator, tumor necrosis factor-α (TNF-α), is secreted during inflammation and is associated with the early onset, as well as long-lasting, inflammation-mediated increase in excitability of peripheral nociceptive neurons. Here we studied the underlying mechanisms of the rapid component of TNF-α-mediated nociceptive hyperexcitability and acute pain hypersensitivity. We showed that TNF-α leads to rapid onset, cyclooxygenase-independent pain hypersensitivity in adult rats. Furthermore, TNF-α rapidly and substantially increases nociceptive excitability in vitro, by decreasing action potential threshold, increasing neuronal gain and decreasing accommodation. We extended on previous studies entailing p38 MAPK-dependent increase in TTX-r sodium currents by showing that TNF-α via p38 MAPK leads to increased availability of TTX-r sodium channels by partial relief of voltage dependence of their slow inactivation, thereby contributing to increase in neuronal gain. Moreover, we showed that TNF-α also in a p38 MAPK-dependent manner increases persistent TTX-r current by shifting the voltage dependence of activation to a hyperpolarized direction, thus producing an increase in inward current at functionally critical subthreshold voltages. Our results suggest that rapid modulation of the gating of TTX-r sodium channels plays a major role in the mediated nociceptive hyperexcitability of TNF-α during acute inflammation and may lead to development of effective treatments for inflammatory pain, without modulating the inflammation-induced healing processes.

Keywords: DRG; inflammatory pain; nociceptor; sodium (Na+) current; tumor necrosis factor.

Copyright © 2015 the American Physiological Society.

Figures

Fig. 1.

Tumor necrosis factor (TNF)-α produces acute cyclooxygenase (COX)-2-independent hypersensitivity to noxious mechanical and thermal stimuli. Latency for paw withdrawal from hot plate (52°C), normalized to baseline (A) and mechanical threshold for paw withdrawal, normalized to baseline, measured by von Frey filaments (B), after intraplantar injection of vehicle, followed, 20 min later, by intraplantar injection of saline (Vehicle-Saline group, black squares); vehicle followed, 20 min later, by intraplantar injection of TNF-α (Vehicle-TNF-α group open circles); COX antagonist, ibuprofen, followed, 20 min later, by intraplantar injection of saline (Ibuprofen-Saline group, gray triangles); or ibuprofen followed by intraplantar injection of TNF-α (Ibuprofen-TNF-α group, light gray inverted triangles). The values are means ± SE; n = 6 for each group. Time point “0” refers to the baseline. Black asterisks, comparison between Vehicle-TNF-α group and Vehicle-Saline group; gray asterisks, comparison between Ibuprofen-TNF-α group and Ibuprofen-Saline group. Nonsignificant (ns), P > 0.05; ***P < 0.001; **P < 0.01; *P < 0.05; two-way ANOVA followed by Bonferroni post-test.

Fig. 2.

Application of TNF-α increases the excitability of nociceptor-like dorsal root ganglion (DRG) neurons. A: representative traces of membrane voltage responses to current steps, recorded from the same small nociceptor-like DRG neurons, before and 10 min after bath application of TNF-α. Note that, following the application of TNF-α, subthreshold stimuli become suprathreshold (Rheobasebefore = 187.3 ± 61.6 pA; RheobaseTNF-α = 139.1 ± 52.8 pA, P < 0.01, n = 11). B: frequency-intensity (f–I) curves calculated from the same neurons, before (gray squares) and 10 min after application of TNF-α (black circles), expressed as spike frequency (means ± SE) evoked by suprathreshold stimulus and plotted against stimuli intensity normalized to rheobase of each analyzed neuron. Note the significantly increased spike frequency (***P < 0.001, two-way ANOVA, n = 10) and increase in slope (m) of the f–I curve following application of TNF-α (P < 0.001, paired t-test, n = 11). C: representative traces of the membrane voltage responses to 1.5-s depolarizing current ramps (333 pA/s) recorded from the same neuron before and after application of TNF-α. Note that TNF-α reduced first spike latency (FSL) and increased action potential (AP) frequency. D: histogram plotting the spike count before and 10 min after application of TNF-α measured from traces as in C. Note the increase in the number of spikes (means ± SE, **P < 0.01, paired t-test, n = 10). E: AP thresholds for generation of APs, obtained by the phase plot analysis of AP, before and 10 min after application of TNF-α (means ± SE, **P < 0.01, paired t-test, n = 11).

Fig. 3.

TNF-α increases the amplitude of tetrodotoxin-resistant (TTX-r) slow and TTX-r persistent sodium currents in nociceptor-like DRG neurons. A–C, left: representative traces of total (A), persistent TTX-r (B) and slow TTX-r (C) sodium currents recorded from the same neuron before (gray traces) and 1 min after application of TNF-α (black traces). The currents were elicited by protocols represented in the inset beneath the traces (for the detailed protocols, see materials and methods). The dashed line indicates the zero current level. Currents are adjusted according to the baseline before the activation step. Traces (150 μs) were blanked to remove residual uncompensated capacitance transient currents. Note the different scale bars between A, B and C. Right: ratio of the peak of total (A), persistent TTX-r (B) and slow TTX-r (C) sodium currents after TNF-α application (Ipeak,TNF-α) to the peak current before (Ipeak,before) from all measured cells (means ± SE, nTotal = 14, nTTX-r per = 5, nTTX-r = 30, paired t-test, ***P < 0.001). Dotted lines indicate the level of Ipeak,before. D: time course of changes of the peak amplitude of TTX-r sodium current assessed by 150-ms depolarizing steps from −70 mV to 0 mV during a 20-min application of TNF-α (black circles) or vehicle (gray squares) (n = 11 for time points −2.5 to 17.5; n = 7 for time point 20; n = 5 for time points 25 to 30; means ± SE). Time point −2.5 refers to data collected shortly after establishing the whole cell configuration when vehicle was applied. Time points 0 to 20 indicate time of application of TNF-α. Note fast onset of the effect of TNF-α (1 min) and decrease in sodium current amplitude when recorded without TNF-α (n = 9 for time points −2.5 to 10; n = 6 for time point 12.5 to 30; n = 5 for time points 25 to 30; means ± SE). Peak current normalized to peak current amplitude detected at −2.5 time point (3.1 ± 1.1 nA for the TNF-α group, 3.2 ± 0.6 nA, for the vehicle group, **P < 0.01, two-way ANOVA, n = 30) is marked by dashed line.

Fig. 4.

Application of TNF-α reduces the threshold and increases the maximal upstroke velocity of the AP, in the presence of TTX. A: representative phase plots of rate of change of the membrane potential (dV/dt) during an AP vs. membrane potential (Vm) recorded from the same neuron before (gray) and 10 min after application of TNF-α (black), in the presence of TTX. Note the leftward shift of the threshold voltage (gray arrows, quantified in B) and increase in maximal velocity, (dV/dt)max, of the upstroke of the AP (black arrows, quantified in C). Inset, traces of APs, before (gray) and after (black) application of TNF-α, which have been used for the generation of the phase plot, presented in A. The arrows indicate the estimated counterpart point presented in the phase plot graph. B: the thresholds for generation of APs, in the presence of TTX, calculated from the same neurons before and 10 min after application of TNF-α (means ± SE, **P < 0.01, paired t-test, n = 8). C: the maximal velocity (dV/dt)max of the upstroke of the AP before (gray) and after (black) application of TNF-α (means ± SE, **P < 0.01, paired t-test, n = 8).

Fig. 5.

TNF-α shifts the activation of TTX-r persistent in hyperpolarized direction. A: averaged peak current-voltage (I–V) characteristics of TTX-r persistent sodium current, before (open squares) and 1 min after application of TNF-α (black circles). To estimate the change in the voltage dependence of activation, we calculated the voltage shift, which entails the minimum “distance” between the sum of control current values [Ibefore (V)] and their approximate equivalents after TNF-α [ITNF-α (V + vstep)]. To this end, Ibefore (V) values were shifted in steps with 0.01-mV increments towards ITNF-α (V), while ITNF-α (V) was kept constant. The shift (arrow) of 10 mV gave the best fit (see materials and methods). Inset: representative traces of TTX-r persistent sodium currents, recorded in the presence of TTX and a blocker of voltage-gated sodium (Nav) 1.8 channels, A803467, elicited by 120-ms steps from a holding potential of −90 mV to −50 mV (light gray), −40 mV (gray) and −30 mV (black) before and 1 min after application of TNF-α. Currents are adjusted according to the baseline before the activation step. The dashed line indicates the zero current level. B: representative instantaneous I–V curve during a slow (36 mV/s) depolarizing ramp from −70 to +20 mV before (gray) and 1 min after application of TNF-α (black). Note “W” shaped inward current with clear early and late components. TNF-α increased the amplitude of both early and late components of the inward current (C) (**P < 0.01, *P < 0.05, paired t-test, n = 6) and shifted the onset (circles) and peak activation (squares) of early, but not the late, component to more negative potentials (D) (***P < 0.001, **P < 0.01, *P > 0.05, paired t-test, n = 6).

Fig. 6.

TNF-α increases TTX-r sodium currents in nociceptor-like DRG neurons by modulating voltage dependence of slow inactivation. A: representative families of TTX-r sodium currents evoked by a series of 150-ms, 10-mV depolarizing voltage steps from a holding potential of −70 mV (top) or −120 mV (bottom), recorded from the same neurons before and 1 min after application of TNF-α. The dashed lines indicate the zero current level. The dotted lines indicate the peak current level before application of TNF-α. B: histogram plotting the ratio of the peak sodium currents 1 min after TNF-α application (Ipeak,TNF-α) to the peak current before application (Ipeak,before) when elicited from a holding potential (Vholding) of −70 mV or −120 mV (means ± SE, ***P < 0.001, **P < 0.01, Student's t-test, n = 30). For A and B, currents are adjusted according to the baseline before the activation step. Note, significant increase in current at holding of −70 mV and significant decrease in current at holding of −120 mV. C: activation [conductance/maximum conductance (G/Gmax)] and availability [current/maximal current (I/Imax)] curves show no change in voltage dependence of activation (P = 0.607, paired t-test between the values of V1/2,before and V1/2,TNF-α, n = 30) and fast inactivation (P = 0.718, paired t-test between the values of V1/2,before and V1/2,TNF-α, n = 14), respectively, following TNF-α. D: superimposed representative traces of TTX-r sodium currents before (open squares) and 3 min after application of TNF-α (closed circles). Currents were elicited by 10-ms test steps to 0 mV, after 5-s conditioning pulses (Vcond) to the indicated voltages. Note that the substantial current reduction, following depolarized Vcond, was diminished by application of TNF-α. Currents are adjusted according to the baseline before the activation step. The dashed line indicates the zero current level. Inset: stimulus protocol (see materials and methods). The values of the current following the test step (Itest) were divided by the maximal current (Imax) and plotted as means ± SE against Vcond to calculate the fraction of channels available for activation at each holding voltage (E) (n = 25). The lines are the best fits to the Boltzmann function (see materials and methods). Note the increase in fraction of available channels following TNF-α [refer to Table 2 for values of the midpoints of voltage dependence (V1/2), values of slope and statistical analysis].

Fig. 7.

In compartmental simulations, rightward shift in voltage dependence of slow inactivation, equivalent to one produced by TNF-α, leads to increase in AP firing rate. A: a Markovian state model for TTX-r sodium channel represents two parallel planes: the first, a classic Hodgkin-Huxley like kinetic scheme with fast voltage-dependent transition; and the second, describing the slow inactivation states with much slower kinetics (see materials and methods). O denotes the open state; C denotes the closed states; IC and SC represent the fast and slow inactivation states, respectively; and SIC is the state in which the channel is inactivated by both slow and fast inactivation mechanisms. Activation occurs with the independent movement of gates, with voltage-dependent transitions between the multiple closed states. B: responses of simulated neuron to 2,000-ms hyperpolarizing and depolarizing suprathreshold current steps without (left) and with (right) 3.32-mV rightward shift in the voltage dependence of slow inactivation of slow Nav 1.8 channels (see materials and methods). A 3.32-mV shift was used to match the shift in voltage dependence in slow inactivation produced by TNF-α (Fig. 6 and Table 2). Note the increase in AP firing following the same stimuli (shown below) when rightward shift is applied. C: f–I curves calculated with (black circles) and without (gray squares) a 3.32-mV rightward shift in the voltage dependence of slow inactivation of Nav 1.8 channels, expressed as spike frequency evoked by suprathreshold stimulus and plotted against stimuli intensity normalized to rheobase. Note the significantly increased spike frequency and increase in slope (m) of the f–I curve following the shift. D: rightward shift of 3.32 mV in voltage dependence of slow inactivation in simulated neuron produced increased firing rate in response to 1.5-s-long depolarizing current ramps (333 pA/s, shown below). E and F: histograms plotting the spike count (E) and the maximal velocity (dV/dt)max of the upstroke of the AP (F) with (black) and without (gray) 3.32-mV depolarizing shift in the voltage dependence of slow inactivation of Nav 1.8 channels.

Fig. 8.

TNF-α-mediated relief of voltage dependence of slow inactivation and increase in nociceptive excitability requires p38 MAPK. A: voltage dependence of slow inactivation of TTX-r sodium currents, expressed as peaks of TTX-r sodium currents during a step to 0 mV, normalized to Imax (fraction available, means ± SE) and plotted against conditioning voltages, before (gray squares) and 2 min after application of p38 MAPK antagonist SB202190, followed by a 1-min coapplication with TNF-α (open circles, paired t-test between the values of V1/2,before and V1/2,TNF-α, P > 0.05, n = 14, see also Table 2). Inset: representative traces of TTX-r sodium currents, recorded in the presence of TTX, elicited by 150-ms steps from a holding potential of −70 mV to 0 mV before (gray) and 2 min after application of p38 MAPK antagonist SB202190, followed by a 1 min coapplication with TNF-α (black). Currents are adjusted according to the baseline before the activation step. The dashed line indicates the zero current level. The dotted line indicates the peak current level before application of TNF-α. B: voltage dependence of slow inactivation of TTX-r sodium currents, assessed as explained in A, before (gray squares) and 2 min after application of the inactive analog, SB202474, followed by a 1-min coapplication with TNF-α (black, paired t-test between the values of V1/2,before and V1/2,TNF-α, P < 0.0001, n = 18; see also Table 2). Inset: representative traces of TTX-r sodium currents, recorded in the presence of 300 nM TTX, elicited by 150-ms steps from a holding potential of −70 mV to 0 mV before (gray) and 2 min after application of the inactive analog SB202424, followed by 1-min coapplication with TNF-α (black). The dashed line indicates the zero current level. The dotted line indicates the peak current level before application of TNF-α. C: the ratio of peak TTX-r sodium current before application of treatment (INa,before) to the peak current after application (INa,treatment) of SB202190 + TNF-α (n = 14) and SB202474 + TNF-α (n = 18). The values are means ± SE; ns, P > 0.05, ***P < 0.001, one-sample t-test comparing the values before and after the application of treatment. D: representative traces of current clamp recordings from the same nociceptor-like DRG neuron, following 500 pA, 1.5-s depolarizing ramps before (left) and 3 min after application of p38 MAPK antagonist SB202190, followed by a 10-min coapplication with TNF-α (right). FSL, first spike latency. E: SB202190 prevents the effect of TNF-α on spike count (means ± SE, **P < 0.01, paired t-test, n = 8) and threshold for generation of AP (F) (means ± SE, ns, P > 0.05, paired t-test, n = 8). G: latency for paw withdrawal from hot plate (5°C), normalized to baseline, after intraplantar injection of vehicle followed, 20 min later, by intraplantar injection of saline (Vehicle-Saline, black squares); intraplantar injection of vehicle followed, 20 min later, by intraplantar injection of TNF-α (Vehicle-TNF-α, open circles); intraplantar injection of p38 MAPK inhibitor, SB203580, followed, 20 min later, by intraplantar injection of saline (SB203580-Saline, gray triangles); or intraplantar injection of SB203580 followed by intraplantar injection of TNF-α (SB203580-TNF-α, light gray inverted triangles). The values are means ± SE. Time point “0” refers to the baseline. Gray asterisks, comparison between SB203580-Saline group to SB203580-TNF-α group. ns, P > 0.05; **P < 0.01; *P < 0.05; two-way ANOVA followed by Bonferroni post-test, n = 6 for each group. H: mechanical threshold for paw withdrawal, normalized to baseline, obtained by von Frey filaments. Time point “0” refers to the baseline. Symbols are as in G.

Fig. 9.

p38 MAPK modulates voltage dependence of slow inactivation. A: voltage dependence of slow inactivation of TTX-r sodium currents, expressed as peaks of TTX-r sodium currents during a step to 0 mV, normalized to Imax (fraction available, means ± SE) and plotted against conditioning voltages before (gray squares) and 3 min after application of SB202190 (open circles, paired t-test, P < 0.05, n = 7. See Table 2 for the values of the V1/2 and slope and statistical analysis). Inset: representative families of TTX-r sodium currents, recorded in the presence of TTX, elicited by 150-ms, 10-mV steps from a holding potential of −70 mV before (gray) and 3 min after application of SB202190 (black). Currents are adjusted according to the baseline before the activation step. The dashed line indicates the zero current level. The dotted line indicates the peak current level before application of SB202190. B: the ratio of peak TTX-r sodium current before application of treatment (INa,before) to the peak current after application (INa,treatment) of vehicle (n = 11) or SB202190 alone (n = 6). Values are means ± SE; **P < 0.01, one sample t-test comparing the values before and after the application of treatment. C: voltage dependence of slow inactivation of TTX-r sodium currents, expressed as peaks of TTX-r sodium currents during steps to 0 mV, normalized to Imax (fraction available, means ± SE) and plotted against conditioning voltages, measured 30 min after pretreatment with the p38 MAPK activator anisomycin (open circles, n = 26) or vehicle (black squares, n = 20), P < 0.01, two-sampled t-test; see also Table 3 for numerical data. Inset: histograms of the distribution of the values of V1/2 of voltage dependence of slow inactivation of TTX-r sodium currents, recorded form neurons preincubated for 30 min with vehicle (gray, n = 20) or 10 μg/ml anisomycin (white, n = 26). Fitting of the distribution with the amplitude version of Gaussian peak function, y = y0 + A × exp(−0.5 × {[V1/2 − V1/2(c)]/w})2, where A is the peak number of cells and V1/2(c) is the value of V1/2 which corresponds to A; black solid line for vehicle; gray solid line for anisomycin, reveals that anisomycin produced substantial rightward shift of population of V1/2 values [V1/2(c),Vehicle = −30.3 ± 1.5 mV; V1/2(c),Anisomycin = −21.2 ± 0.9 mV]. D: the peak TTX-r current density, measured from currents elicited by depolarizing steps to 0 mV from a holding potential of −70 mV, in cells pretreated with anisomycin or vehicle. Values are means ± SE, *P < 0.01, two sampled t-test, n = 26 for anisomycin group, n = 20 for vehicle group.

Fig. 10.

Pharmacological enhancement of voltage dependence of slow inactivation reverses the TNF-α-mediated relief of slow inactivation and diminishes the effect of TNF-α on nociceptive excitability. A, left: voltage dependence of slow inactivation of TTX-r sodium currents, expressed as peaks of TTX-r sodium currents during a step to 0 mV, normalized to Imax (fraction available, means ± SE) and plotted against conditioning voltages before (gray squares) and after application of TNF-α, together with lacosamide (open circles). Note: there was no change in voltage dependence of slow inactivation (see Table 2 for V1/2,slope values and statistical analysis, n = 8). A, right: the ratio of peak TTX-r sodium current before application of treatment (INa,control) to the peak current after the treatment (INa,treatment) with lacosamide and lacosamide + TNF-α. Values are means ± SE, n = 10; P = 0.32, compared with the control, one-sample t-test. B, left: voltage dependence of slow inactivation of TTX-r sodium currents, before (gray squares) and after application of TNF-α, together with brilliant blue G (BBG) (open circles). Note: there was no change in voltage dependence of slow inactivation (see Table 2 for V1/2,slope values and statistical analysis, n = 4). B, right: the ratio of peak TTX-r sodium current before application of treatment (INa,control) to the peak current after the treatment (INa,treatment) with BBG and BBG + TNF-α. Values are means ± SE, n = 4, P = 0.6, compared with the control, one-sample t-test. C, top: representative traces of current clamp recordings from the same nociceptor-like DRG neuron, following 500 pA, 1.5-s depolarizing ramps in control condition (left trace), 10 min after application of TNF-α (middle trace) and 5 min after application of lacosamide on top of 10 min application of TNF-α (right trace). See Table 4 for the numerical data and statistical analysis. Bottom: stimulation protocol. D: latency for paw withdrawal from hot plate (52°C), normalized to baseline, after intraplantar injection of saline (black squares); TNF-α alone (open circles); lacosamide alone (gray triangles); or TNF-α applied together with lacosamide (light gray inverted triangles). The values are means ± SE. Time point “0” refers to the baseline. Gray asterisks, comparison between the TNF-α group to TNF-α + Lacosamide group; ns, P > 0.05, *P < 0.05, two-way ANOVA followed by Bonferroni posttest, n = 6 for each group. E: mechanical threshold for paw withdrawal, normalized to baseline, obtained by von Frey filaments. The values are means ± SE, n = 6 for each group. Time point “0” refers to the baseline. Symbols are as in E; **P < 0.01.

Fig. 11.

TNF-α relieves TTX-r channels from ultraslow inactivation at hyperpolarized potentials. A fraction of channels available for activation (peaks of TTX-r sodium currents during a step to 0 mV, normalized to Imax, means ± SE) following prolonged 30-s holding at voltages, as indicated in the x-axis, before (gray squares) and 3 min after application of TNF-α (black circles). Currents were elicited by 10-ms test steps to 0 mV, after 30-s conditioning pulses (Vcond) to the indicated voltages. Note that the substantial current reduction, following prolonged holding at Vcond of −70 mV, −60 mV and −50 mV (black asterisks, comparison within Before group, ns, P > 0.05, **P < 0.01, ***P < 0.001 one-way ANOVA with post hoc Bonferroni, n = 6), was diminished by application of TNF-α (gray asterisks, comparison within TNF-α group, ns, P > 0.05, ***P < 0.001, one-way ANOVA with post hoc Bonferroni, n = 6). Note also that TNF-α leads to increase of available channels at −70 mV by about 15%. Light great asterisks, comparison between Before and TNF-α groups. *P < 0.05; **P < 0.01, two-way ANOVA with post hoc Bonferroni, n = 6. Inset: stimulation protocol.

Fig. 12.

Scheme depicting TNF-α-mediated nociceptive hyperexcitability. Left: in normal conditions, only a fraction of Nav 1.8 channels, which underlie slow TTX-r sodium current, are available for activation, due to slow inactivation (denoted by collapsed outer pore of the channel). This, together with the voltage dependence of activation of Nav 1.9 channels, which underlies persistent TTX-r sodium current, defines a threshold for AP initiation and the ability to fire repetitively after threshold is achieved. TNFR, TNF receptor. Right: TNF-α binds to its receptor and, via p38 MAPK, leads to a hyperpolarization shift in the activation of Nav 1.9 channels. Moreover, TNF-α-mediated activation of p38 MAPK, probably via direct phosphorylation of Nav 1.8 channels (see Hudmond et al. 2008 and also Fig. 9 here) increases the availability of Nav 1.8 channels, by modulating their slow inactivation. These processes lead to decrease in threshold, such that previously subthreshold stimuli now evoke APs, and to increase in gain, enhancing the ability of nociceptive neurons to fire repetitively, altogether producing nociceptive hyperexcitability.