This work was motivated by the question of how AP conduction can be maintained in the central projections of nociceptive DRG neurons which course through the dorsal horn where they are subjected to depolarization [
1,
2] and by the observations that Na
v1.8 channels are present within the central terminals of primary sensory neurons within the dorsal horn [
23,
24] and that the amplitude of APs recorded from small DRG neurons is dependent on the holding potential of the cell, and that this voltage dependence is best fit with two Boltzmann equations. Because sodium channels provide the primary source of current for the AP upstroke, the latter finding suggested a method for investigating the contribution of the various sodium channels expressed by small DRG neurons to AP generation. The V
1/2 values for the two Boltzmann fits (-73 mV and 37 mV)are similar to the V
1/2 values for fast steady-state inactivation associated with TTX-s channels (-65 to -75 mV) and Na
v1.8 TTX-r channels (-30 to -40 mV), respectively, that are expressed in small DRG neurons [
6,
7,
9‐
13]. The substantial contribution of the component with the more depolarized V
1/2 to AP amplitude (81%), is consistent with previous estimates of the relative contribution of Na
v1.8 current to AP amplitude. Using the Goldman-Hodgkin-Katz equation to estimate sodium ion permeability during the AP in DRG from Na
v1.8(+/+) and Na
v1.8(-/-) animals, Renganathan concluded that Na
v1.8 channels contribute 80–90% of the current that flows at the peak of the AP [
20]. Blair and Bean isolated TTX-s, TTX-r and high voltage-activated calcium currents in small DRG neurons stimulated with a simulated AP voltage protocol [
21]. They estimated that TTX-r currents contributed 60% of the AP upstroke, as compared to 40% for the TTX-s component.
To confirm the contribution of TTX-s channels, we determined the voltage-dependence of AP amplitude in the presence of 300 nM TTX. For V
h values of approximately -60 to -20 mV, AP amplitude decreased gradually, and this voltage-dependence was well fit with a single Boltzmann equation having a V
1/2 of -36 mV. The absence of the more hyperpolarized component demonstrates a contribution of TTX-s sodium currents. In place of a decrease in AP amplitude, we observed that AP amplitude increased for Vh values of -90 to -60 mV. It is unlikely that an increasing availability of TTX-r channels could account for increasing AP amplitude in this voltage range because it does not fall with the range for steady-state fast inactivation of these channels. Alternatively, we hypothesized that outward current generated by potassium channel opening might be responsible for the change in AP amplitude in the voltage range. To test this hypothesis, we recorded AP amplitude under conditions known to block the majority of voltage-gated potassium channels (TEA and 4-AP). Estimates of outward current amplitude obtained before switching to current clamp mode (see Methods) indicated that 80–90% of the outward current produced by depolarizing voltage steps was blocked. In current clamp mode, analysis of AP amplitude and V
h revealed that the 45% increase in AP amplitude from -90 to -60 mV in the presence of TTX alone decreased to only 9% in the presence of TEA and 4-AP, representing a reduction of 80%. Although we cannot state with certainty the identity of the potassium channels reducing AP amplitude from -90 to -60 mV, possible candidates are the inwardly rectifying current I
IR [
25] and I
h [
26], a slowly activated inward current, both of which are initiated by membrane hyperpolarization. It is interesting to consider the possibility that one of the roles of TTX-s sodium currents in small DRG neurons is to boost AP amplitude at these hyperpolarized membrane potentials.
In the absence of well-established blockers of the Na
v1.8 sodium current, we used DRG neurons from Na
v1.8(-/-) animals to further investigate the identity of the currents contributing to the depolarized V
1/2 characterizing AP amplitude reduction. Previous studies have demonstrated that small DRG neurons from these animals completely lack the slow TTX-r current produced by Na
v1.8 channels [
27]. AP amplitude in these cells decreased with a voltage dependence that was well fit by a single Boltzmann equation with a V
1/2 of -55 mV. This value represents a depolarizing shift of 20 mV compared to the V
1/2 for the TTX-s component in WT neurons. One possible explanation is that the inactivation properties of the remaining TTX-r sodium channels, Na
v1.9, are combining with those of TTX-s sodium channels to produce a single depolarized V
1/2. The midpoint of steady-state fast inactivation for Na
v1.9 in DRG cells ranges from -44 to -54 mV [
14,
28]. However, the slow onset of Na
v1.9 channel openings indicates that these channels make only a minor contribution to AP amplitude [
22,
28]. Akopian et al [
27] observed an up-regulation of Na
v1.7 in Na
v1.8(-/-) DRG neurons; however, this would not account for the shift in V
1/2 because the V
1/2 for steady-state fast inactivation of Na
v1.7, -71 mV to -78 mV [
15,
29], is similar to the more hyperpolarized V
1/2 associated with the TTX-s component of AP decrease in WT neurons. An alternative explanation for the shift in the V
1/2 of the TTX-s component is that the inactivation properties of TTX-s sodium channels are modified in DRG neurons of Na
v1.8(-/-) animals. Such a modification has been observed in a previous study [
20]. In a comparison of TTX-s currents in small DRG neurons from Na
v1.8(+/+) and Na
v1.8(-/-) mice, the authors observed a 20 mV depolarizing shift in the voltage dependence of fast inactivation. The mechanism for this shift in TTX-s voltage dependence of inactivation was not determined. Possible mechanisms include G-protein activation, which has been shown to depolarize inactivation V
1/2 of Na
v1.8 currents by 3–4 mV in DRG neurons [
30], the presence of arachidonic acid, which hyperpolarizes the inactivation V
1/2 of both TTX-s and TTX-r currents in DRG neurons [
31] and tyrosine kinase phosphorylation, which has been shown to both depolarize the V
1/2 of fast inactivation in cardiac Na
v1.5 channels in HEK293 cells, [
32] and hyperpolarize the V
1/2 of fast inactivation in sodium currents of differentiated PC-12 cells [
33]. Because expression of β subunits can influence sodium channel inactivation properties [
34], differential expression of β subunits between Na
v1.8(+/+) and Na
v1.8(-/-) animals might also account for the observed shift. Irrespective of the modulatory process involved, our additional results with transfection of Na
v1.8 into cells from Na
v1.8(-/-) animals demonstrates that the process is not permanent and is dependent on the absence of Na
v1.8 channels. After transfections, AP amplitude dependence on V
h was again fit best by two Boltzmann equations in half of the cells, and the V
1/2 values (-71 and -36 mV) were similar to values for the wild type DRG neurons. These results indicate that the reintroduced Na
v1.8 channels led to a shift in the inactivation voltage-dependence of the TTX-s component back to the WT value.