Tetrodotoxin-resistant sodium channels in sensory neurons generate slow resurgent currents that are enhanced by inflammatory mediators

Abstract

Resurgent sodium currents contribute to the regeneration of action potentials and enhanced neuronal excitability. Tetrodotoxin-sensitive (TTX-S) resurgent currents have been described in many different neuron populations, including cerebellar and dorsal root ganglia (DRG) neurons. In most cases, sodium channel Nav1.6 is the major contributor to these TTX-S resurgent currents. Here we report a novel TTX-resistant (TTX-R) resurgent current recorded from rat DRG neurons. The TTX-R resurgent currents are similar to classic TTX-S resurgent currents in many respects, but not all. As with TTX-S resurgent currents, they are activated by membrane repolarization, inhibited by lidocaine, and enhanced by a peptide-mimetic of the β4 sodium channel subunit intracellular domain. However, the TTX-R resurgent currents exhibit much slower kinetics, occur at more depolarized voltages, and are sensitive to the Nav1.8 blocker A803467. Moreover, coimmunoprecipitation experiments from rat DRG lysates indicate the endogenous sodium channel β4 subunits associate with Nav1.8 in DRG neurons. These results suggest that slow TTX-R resurgent currents in DRG neurons are mediated by Nav1.8 and are generated by the same mechanism underlying TTX-S resurgent currents. We also show that both TTX-S and TTX-R resurgent currents in DRG neurons are enhanced by inflammatory mediators. Furthermore, the β4 peptide increased excitability of small DRG neurons in the presence of TTX. We propose that these slow TTX-R resurgent currents contribute to the membrane excitability of nociceptive DRG neurons under normal conditions and that enhancement of both types of resurgent currents by inflammatory mediators could contribute to sensory neuronal hyperexcitability associated with inflammatory pain.

Keywords: action potential; hyperexcitability; nociceptor; resurgent sodium current; sodium current; voltage clamp.

Figure 1.

DRG neurons exhibit TTX-R and TTX-S resurgent currents. A, B, Representative TTX-R and TTX-S sodium currents were recorded from medium-sized DRG neurons (30–45 μm) using a voltage-protocol (as shown at bottom) designed for recording resurgent sodium currents. TTX-R resurgent currents were recorded in the presence of 500 nm TTX. The repolarization voltages for colored current traces are listed according to the color and relative top-down position of colored traces. The peak resurgent current traces are black. The voltages for peak resurgent currents are labeled nearby. C, Current–voltage relationship of TTX-R and TTX-S resurgent currents. The peak voltage of TTX-R resurgent currents is more depolarized than that of TTX-S resurgent currents. D, Effects of lidocaine (n = 5), CsCl (n = 5), and A803467 (1 μm, n = 7; 5 μm, n = 6) on TTX-R resurgent currents. The peak current amplitude in the presence of each compound was normalized to its own control. Data are mean ± SEM. **p < 0.01 (Student's t test).

Figure 2.

Association of NaV1.8 with β4 subunits in DRG neurons. A, Immunoblot results from the coimmunoprecipitation experiment from rat brain lysate. Top, Sodium channel α subunit bands (200–260 kDa) are observed in the lysate input indicating that CNS NaV protein is present in the IP input lysate. Bottom, Sodium channel β4 subunit protein band is observed in the lysate input indicating that β4 subunit protein was present in the IP input lysate. Right, Neither NaV1.8 channel nor β4 subunit protein was coimmunoprecipitated from the rat brain (rBrain) lysate matrix using either the control IgG(Rb) or NaV1.8 antibody. B, Immunoblot results from the coimmunoprecipitation experiment from rat DRG lysate. Top, Sodium channel α subunit bands (200–260 kDa) are observed in the lysate input indicating that peripheral NaV protein is present in the IP input lysate. Bottom, β4 subunit protein band is observed in the lysate input indicating that β4 subunit protein was present in the IP input lysate. Right, NaV1.8 channel and β4 subunit protein are coimmunoprecipitated from rat DRG (rDRG) lysate only by the anti-NaV1.8 Asc-016 antibody.

Figure 3.

Increase of TTX-R and TTX-S resurgent currents by a β4 peptide in DRG neurons. Representative TTX-R (A, B) and TTX-S (C, D) resurgent currents were recorded from medium-sized DRG neurons in the absence and presence of 200 μm β4 peptide in the patch pipettes. The currents were recorded using the same voltage protocol in Fig. 1. Currents are scaled to reflect the ratio resurgent currents. The ratio resurgent currents were calculated by normalizing peak resurgent currents to peak transient currents recorded in the same cells. E, F, Summary of ratio resurgent current and voltage at which peak resurgent currents were recorded (TTX-R control, n = 16; TTX-R plus peptide, n = 8; TTX-S control, n = 12; TTX-S plus peptide, n = 6). White bars represent control; black bars represent plus peptide. Data are mean ± SEM. *p < 0.05 (Student's t test). **p < 0.01 (Student's t test).

Figure 4.

Simulations of TTX-R and TTX-S resurgent currents. A, Comparison of TTX-R (red trace) and TTX-S (black trace) resurgent currents recorded from DRG neurons. Traces were elicited with a −20 mV repolarization step after a 20 ms depolarization to 30 mV. B, Comparison of simulated TTX-S (black trace) and TTX-R (red trace) transient currents elicited with a step depolarization to −20 mV from a holding potential of −100 mV. C, Comparison of simulated TTX-S (black trace) and TTX-R (red trace) resurgent currents elicited with a −20 mV repolarization step after a 20 ms depolarization to 30 mV. D, Current–voltage relationship of simulated TTX-R and TTX-S resurgent currents. The peak voltage of TTX-R resurgent currents is more depolarized than that of TTX-S resurgent currents. E, Increasing the time constant of TTX-S inactivation slows the decay of simulated resurgent currents. Resurgent currents generated with control TTX-S model (black trace) and TTX-S channels with fivefold (blue trace) and 10-fold (green trace) longer time constants for inactivation are shown. F, Reducing the rate constant for exit from open-channel block by fivefold (blue trace) decreased both the onset of TTX-R resurgent current and the decay time constant for the resurgent current.

Figure 5.

Increase of TTX-R resurgent currents by inflammatory mediators (IMs) in DRG neurons. Representative TTX-R (A, B) and TTX-S (C, D) resurgent currents were recorded from medium-sized DRG neurons in the absence and presence of IMs. IMs (1 μm bradykinin, 10 μm 5-HT, 10 μm histamine, 10 μm PGE₂, and 5 μm ATP) were pretreated for 5 min in the recording chamber before recording begins. The currents were recorded using the same voltage protocol in Fig. 1. Currents are scaled to reflect the ratio resurgent currents. The ratio resurgent currents were calculated by normalizing peak resurgent currents to peak transient currents recorded in the same cells. E, F, Summary of ratio resurgent current and voltage at which peak resurgent currents were recorded (TTX-R control n = 14; TTX-R plus IMs n = 10; TTX-S control n = 12; TTX-S plus IMs n = 19). White bars represent control; black bars represent plus IMs. Data are mean ± SEM. *p < 0.05 (Student's t test).

Figure 6.

Increase of TTX-R excitability in small DRG neurons by a β4 peptide. Small DRG neurons were examined under current-clamp conditions; 500 nm TTX was included in the bath solution to block TTX-S sodium channels. A series of depolarizing current steps (0 pA up to 3× rheobase with a 100 pA increment) were injected into small DRG neurons from their resting membrane potentials. Representative membrane responses to current injection from 0 pA to rheobase, 2× rheobase, and 3× rheobase were shown in control (A) and 200 μm β4 peptide (B). Compared with control, β4 peptide significantly (p < 0.01, χ² test) increased the percentage of small DRG neurons that fire multiple action potentials after suprathreshold current injection up to 3× rheobase (C).