Inactivation properties of sodium channel Nav1.8 maintain action potential amplitude in small DRG neurons in the context of depolarization

Abstract

Background: Small neurons of the dorsal root ganglion (DRG) express five of the nine known voltage-gated sodium channels. Each channel has unique biophysical characteristics which determine how it contributes to the generation of action potentials (AP). To better understand how AP amplitude is maintained in nociceptive DRG neurons and their centrally projecting axons, which are subjected to depolarization within the dorsal horn, we investigated the dependence of AP amplitude on membrane potential, and how that dependence is altered by the presence or absence of sodium channel Nav1.8.

Results: In small neurons cultured from wild type (WT) adult mouse DRG, AP amplitude decreases as the membrane potential is depolarized from -90 mV to -30 mV. The decrease in amplitude is best fit by two Boltzmann equations, having V1/2 values of -73 and -37 mV. These values are similar to the V1/2 values for steady-state fast inactivation of tetrodotoxin-sensitive (TTX-s) sodium channels, and the tetrodotoxin-resistant (TTX-r) Nav1.8 sodium channel, respectively. Addition of TTX eliminates the more hyperpolarized V1/2 component and leads to increasing AP amplitude for holding potentials of -90 to -60 mV. This increase is substantially reduced by the addition of potassium channel blockers. In neurons from Nav1.8(-/-) mice, the voltage-dependent decrease in AP amplitude is characterized by a single Boltzmann equation with a V1/2 value of -55 mV, suggesting a shift in the steady-state fast inactivation properties of TTX-s sodium channels. Transfection of Nav1.8(-/-) DRG neurons with DNA encoding Nav1.8 results in a membrane potential-dependent decrease in AP amplitude that recapitulates WT properties.

Conclusion: We conclude that the presence of Nav1.8 allows AP amplitude to be maintained in DRG neurons and their centrally projecting axons even when depolarized within the dorsal horn.

Figure 1

The voltage dependent decrease in AP amplitude recorded from a small diameter, WT DRG neuron is best fit by 2 Boltzmann equations. APs were elicited by threshold stimulation while holding the cell membrane potential between -90 and -25 mV. Inset waveforms on the right represent responses elicited at the membrane holding potential indicated by the small letters (a-f). The dotted lines in the insets indicate 0 mV. The solid and dashed lines in the main figure represent Boltzmann equation fits with V_1/2 values of -74.3 mV and -34.7 mV, respectively. For this cell, the contribution of the more hyperpolarized component accounted for 10% of the decrease in AP amplitude.

Figure 2

Block of TTX-s sodium channels alters the voltage dependent decrease in AP amplitude. Responses from a single small DRG neuron in the presence of 300 nM TTX (inset waveforms a-f on the right) demonstrate that AP amplitude increases with depolarization of membrane holding potential from -90 to -60 mV (inset waveforms a-c). From holding potentials of -60 to -25, AP amplitude decreases with depolarization (inset waveforms c-f), and this decrease can be fit with a single Boltzmann equation (dashed line in the main figure, V_1/2 = -36.8 mV). The dotted lines in inset waveforms indicate 0 mV.

Figure 3

Potassium channel blockers reduce outward currents. Voltage clamped currents in response to depolarizing voltage steps from a holding potential of -100 mV are shown for two separate small DRG neurons. Cells were recorded in the absence (top) and presence (bottom) of the potassium channel blockers TEA and 4-AP.

Figure 4

Potassium channel blockers reduce the increase in AP amplitude observed in the presence of TTX for hyperpolarized holding potentials. Responses from a single small DRG neuron (inset waveforms a-f on the right) demonstrate that the increase in AP amplitude observed in the presence of TTX with depolarization of membrane holding potential from -90 to -60 mV (inset waveforms a-c) is much smaller in the presence of TEA and 4-AP. In contrast, the decrease in AP amplitude with depolarization from holding potentials of -60 to -25 mV (inset waveforms c-f) is unchanged in the presence of TEA and 4-AP. The decrease can be fit with a single Boltzmann equation (dashed line in main figure, V_1/2 = -38.8 mV). The dotted lines in inset waveforms indicate 0 mV.

Figure 5

The absence of Na_v1.8 sodium channels alters the voltage dependent decrease in AP amplitude. Responses from a small DRG neuron cultured from a Na_v1.8(-/-) mouse are shown as inset waveforms on the right (a-f). In these neurons, the decrease in AP amplitude as membrane holding potential is depolarized from -90 to -30 mV is best fit by a single Boltzmann equation (solid line in main figure). The V_1/2 (-53.4 mV for this cell) is about 20 mV more depolarized than the TTX-s component in small DRG neurons from WT mice. The dotted lines in inset waveforms indicate 0 mV.

Figure 6

Transfection with Na_v1.8 produces two populations of cells with respect to voltage-dependence of AP amplitude. Data are from two small DRG neurons from Na_v1.8(-/-) mice transfected with Nav1.8. The cell in (A) is representative of a population of cells (N = 7) for which the decrease in AP amplitude was best fit with single Boltzmann equation having a V_1/2 of -53.9 mV (solid line). The cell in (B) is representative of a population of cells (N = 8) for which the decrease in AP amplitude was best fit by two Boltzmann equations, with V_1/2 values of -72.6 mV (solid line) and -33.3 mV (dashed line).