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. 2020 Jul 1;124(1):274-283.
doi: 10.1152/jn.00188.2020. Epub 2020 Jun 10.

IL-6 induced upregulation of T-type Ca2+ currents and sensitization of DRG nociceptors is attenuated by MNK inhibition

Affiliations

IL-6 induced upregulation of T-type Ca2+ currents and sensitization of DRG nociceptors is attenuated by MNK inhibition

Vivek Jeevakumar et al. J Neurophysiol. .

Abstract

Phosphorylation of the 5' cap-binding protein eIF4E by MAPK-interacting kinases (MNK1/2) is important for nociceptor sensitization and the development of chronic pain. IL-6-induced dorsal root ganglion (DRG) nociceptor excitability is attenuated in mice lacking eIF4E phosphorylation, in MNK1/2-/- mice, and by the nonselective MNK1/2 inhibitor cercosporamide. Here, we sought to better understand the neurophysiological mechanisms underlying how IL-6 causes nociceptor excitability via MNK-eIF4E signaling using the highly selective MNK inhibitor eFT508. DRG neurons were cultured from male and female ICR mice, 4-7 wk old. DRG cultures were treated with vehicle, IL-6, eFT508 (pretreat) followed by IL-6, or eFT508 alone. Whole cell patch-clamp recordings were done on small-diameter neurons (20-30 pF) to measure membrane excitability in response to ramp depolarization. IL-6 treatment (1 h) resulted in increased action potential firing compared with vehicle at all ramp intensities, an effect that was blocked by pretreatment with eFT508. Basic membrane properties, including resting membrane potential, input resistance, and rheobase, were similar across groups. Latency to the first action potential in the ramp protocol was lower in the IL-6 group and rescued by eFT508 pretreatment. We also found that the amplitudes of T-type voltage-gated calcium channels (VGCCs) were increased in the DRG following IL-6 treatment, but not in the eFT508 cotreatment group. Our findings are consistent with a model wherein MNK-eIF4E signaling controls the translation of signaling factors that regulate T-type VGCCs in response to IL-6 treatment. Inhibition of MNK with eFT508 disrupts these events, thereby preventing nociceptor hyperexcitability.NEW & NOTEWORTHY In this study, we show that the MNK inhibitor and anti-tumor agent eFT508 (tomivosertib) is effective in attenuating IL-6 induced sensitization of dorsal root ganglion (DRG) nociceptors. Pretreatment with eFT508 in DRG cultures from mice helps mitigate the development of hyperexcitability in response to IL-6. Furthermore, our data reveal that the upregulation of T-type voltage-gated calcium channels following IL-6 application can be blocked by eFT508, implicating the MNK-eIF4E signaling pathway in membrane trafficking of ion channels.

Keywords: Cav3.2; DRG excitability; MNK; eFT508; interleukin-6.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

Fig. 1.
Fig. 1.
MAPK-interacting kinases (MNK1/2) inhibition by eFT508 prevents IL-6-induced hyperexcitability in dorsal root ganglion (DRG) nociceptors. A: representative traces of ramp current-induced action potential firing, with ramp stimuli shown at bottom. B: for each ramp intensity tested, the IL-6 group had the highest number of action potentials (AP). The increase in spike firing was prevented by pretreatment with eFT508. No differences were seen in eFT508-treated group alone. *P < 0.05, post hoc Dunnett’s test following 2-way ANOVA. Vehicle vs. IL-6 at 100 pA: mean difference −2.93 [95% confidence interval (95CI) −4.93, −0.92]; at 300 pA: −5.43 [95CI −8.87, −1.99]; at 500 pA: −6.57 [95CI −10.87, −2.27]; at 700 pA: −6 [95 CI −10.75, −1.25]. In the vehicle vs. eFT508+IL-6 and vehicle vs. eFT508, P > 0.05. C: representative traces of single action potentials in all groups evoked at rheobase current using a step protocol. Number of cells: vehicle, 14 from 8 mice; IL-6, 14 from 9 mice; eFT508+IL-6, 12 from 4 mice; eFT508, 10 from 6 mice.
Fig. 2.
Fig. 2.
MAPK-interacting kinases (MNK1/2) inhibition by eFT508 prevents IL-6-induced shift in latency to the action potential (AP) peak in dorsal root ganglion (DRG) nociceptors. A: latency to the first spike measured from the initiation of ramp until the peak of the first action potential was lowered in the IL-6 group. Pretreatment with eFT508 prevented this reduction in latency. No change was observed in the eFT508 treatment group. B: a representative trace indicating the measurement of latency. *P < 0.05, post hoc Dunnett’s test following 2-way ANOVA. Vehicle vs. IL-6 at 100 pA: mean difference 416.9 [95% confidence interval (95CI) 179, 654.8]; at 300 pA: 279.8 [95CI 90.18, 469.5]; at 500 pA: 236.1 [95CI 33.99, 438.2]; at 700 pA: 200 [95CI 12.53, 387.4]. Number of cells: vehicle, 14 from 8 mice; IL-6, 14 from 9 mice; eFT508+IL-6, 12 from 4 mice; eFT508, 10 from 6 mice.
Fig. 3.
Fig. 3.
IL-6 treatment increases T-type calcium currents, and eFT508 pretreatment prevents this upregulation. A: representative traces of inward T-type calcium currents evoked from a holding potential of −90 mV and stepping from −60 mV to 0 mV in 5-mV increments. B: representative traces from all treatment groups, from a holding potential of −90 mV and test potential of 0 mV, showing that IL-6 treatment increases the amplitude of T-type currents. C: current-voltage (I-V) curves of the calcium current density (pA/pF). *P < 0.05, post hoc Dunnett’s test following 2-way ANOVA. Vehicle vs. IL-6 at −10 mV: mean difference 1.434 [95% confidence interval (95CI) 0.33, 2.54]; at −5 mV: 1.3 [95CI 0.19, 2.41]; at 0 mV: 1.8 [95CI 0.68, 2.91]. D: I-V curves of the current amplitudes used to obtain the density in C. *P < 0.05, post hoc Dunnett’s test following 2-way ANOVA. Vehicle vs. IL-6 at −15 mV: mean difference 32.59 [95% confidence interval (95CI) 5.06, 60.12]; at −10 mV: 44.17 [95CI 16.64, 71.7]; at −5 mV: 41.96 [95CI 14.44, 69.49]; at 0 mV: 52.81 [25.28, 80.34]. Inset shows the activation curve G/Gmax. The curve was obtained by fitting the conductances (G) of the amplitudes from D and estimated reversal potential (Erev) of 60 mV to the equation G/Gmax = 1/{1 + exp[(V50V)/slope]}, where V50 is the voltage at which current is half-maximal and V is the step voltage. E: average amplitudes of a subset of cells from D showing the effect of application of TTA-P2 on T-type calcium currents. TTA-P2 application depresses the amplitude of T-type currents. Data are shown for the vehicle, IL-6, and eFT508+IL-6 groups. *P < 0.05, main effect of treatment, repeated measures ANOVA. Number of cells: vehicle, 10 from 5 mice; IL-6, 11 from 6 mice; eFT508+IL-6, 8 from 2 mice; eFT508, 7 from 3 mice.
Fig. 4.
Fig. 4.
T-type calcium channel antagonist TTA-P2 modifies IL-6-induced changes in membrane properties of dorsal root ganglion (DRG) neurons. A: TTA-P2 does not alter the number of action potentials (AP) in IL-6-treated cells in response to ramp currents. B: following 1 h of IL-6 treatment, the latency to the first spike is decreased, and this is reversed following the application of TTA-P2 at 100-pA and 300-pA ramp intensities. Inset shows representative traces from the same cell before and after TTA-P2 application indicating increased latency at 100 pA. *P < 0.05, post hoc Bonferroni correction following 2-way ANOVA. Mean difference at 100 pA: −170.3 [95% confidence interval (95CI) −224.9, −115.6]; at 300 pA: −64.07 [95CI −118.7, −9.41]. C, left: the rheobase to evoke an action potential is increased following TTA-P2 application. Right, representative traces from the same IL-6-treated cell before and after TTA-P2 application in response to a 5-s step protocol. The current required to evoke an action potential increases from 220 pA to 250 pA after TTA-P2 application. *P < 0.05, paired t test. Mean difference: 31.67 [95CI 17.63, 45.7]. Number of cells: 13 from 3 mice.

Comment in

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