Ion channel regulation and function (14)
Pain Mechanisms and Treatments (10)
Neuroscience and Neuropharmacology Research (5)
Ion Channels and Receptors (4)
Cardiac electrophysiology and arrhythmias (3)
Proceedings of the National Academy of Sciences of the United States of America •
Voltage-gated sodium channel Nav1.8 plays a crucial role in regulating excitability of small dorsal root ganglion (DRG) neurons and is an emerging target for pain therapeutics. Using dynamic clamp, we systematically manipulated Nav1.8 conductance to assess its impact on action potential (AP) electrogenesis, rheobase, and repetitive firing in native rat DRG neurons and those expressing the gain-of-function Nav1.7L858H mutation which underlies inherited erythromelalgia, a human genetic pain disorder. Our findings reveal that the Nav1.8 contribution to net sodium current is highly correlated with AP voltage threshold. Nav1.8 conductance regulated AP overshoot and voltage threshold without significantly affecting undershoot or resting membrane potential. We identified two populations of wild-type DRG neurons: strong responders (50% of cells), which exhibited substantial rheobase modulation with alterations in Nav1.8 conductance, and weak responders (50% of cells), which remained largely unaffected. In hyperexcitable Nav1.7L858H-expressing neurons, partial Nav1.8 subtraction (50%) restored rheobase above control levels in 63% of cells. However, weak responders (37%) remained hyperexcitable. The effect of Nav1.8 subtraction in responsive neurons supports the conclusion that Nav1.8 inhibition can reduce neuropathic pain. However, the presence of weakly responsive DRG neurons suggests that other channels might need to be targeted for full pain relief.
While voltage-gated sodium channels Nav1.7 and Nav1.8 both contribute to electrogenesis in dorsal root ganglion (DRG) neurons, details of their interactions have remained unexplored. Here, we studied the functional contribution of Nav1.8 in DRG neurons using a dynamic clamp to express Nav1.7L848H, a gain-of-function Nav1.7 mutation that causes inherited erythromelalgia (IEM), a human genetic model of neuropathic pain, and demonstrate a profound functional interaction of Nav1.8 with Nav1.7 close to the threshold for AP generation. At the voltage threshold of -21.9 mV, we observed that Nav1.8 channel open-probability exceeded Nav1.7WT channel open-probability ninefold. Using a kinetic model of Nav1.8, we showed that a reduction of Nav1.8 current by even 25-50% increases rheobase and reduces firing probability in small DRG neurons expressing Nav1.7L848H. Nav1.8 subtraction also reduces the amplitudes of subthreshold membrane potential oscillations in these cells. Our results show that within DRG neurons that express peripheral sodium channel Nav1.7, the Nav1.8 channel amplifies excitability at a broad range of membrane voltages with a predominant effect close to the AP voltage threshold, while Nav1.7 plays a major role at voltages closer to resting membrane potential. Our data show that dynamic-clamp reduction of Nav1.8 conductance by 25-50% can reverse hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes pain in humans and suggests, more generally, that full inhibition of Nav1.8 may not be required for relief of pain due to DRG neuron hyperexcitability.
We show here that hyperpolarization-activated current (I ) unexpectedly acts to inhibit the activity of dorsal root ganglion (DRG) neurons expressing WT Nav1.7, the largest inward current and primary driver of DRG neuronal firing, and hyperexcitable DRG neurons expressing a gain-of-function Nav1.7 mutation that causes inherited erythromelalgia (IEM), a human genetic model of neuropathic pain. In this study we created a kinetic model of I and used it, in combination with dynamic-clamp, to study I function in DRG neurons. We show, for the first time, that I increases rheobase and reduces the firing probability in small DRG neurons, and demonstrate that the amplitude of subthreshold oscillations is reduced by I . Our results show that I , due to slow gating, is not deactivated during action potentials (APs) and has a striking damping action, which reverses from depolarizing to hyperpolarizing, close to the threshold for AP generation. Moreover, we show that I reverses the hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes IEM. In the aggregate, our results show that I unexpectedly has strikingly different effects in DRG neurons as compared to previously- and well-studied cardiac cells. Within DRG neurons where Nav1.7 is present, I reduces depolarizing sodium current inflow due to enhancement of Nav1.7 channel fast inactivation and creates additional damping action by reversal of I direction from depolarizing to hyperpolarizing close to the threshold for AP generation. These actions of I limit the firing of DRG neurons expressing WT Nav1.7 and reverse the hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes IEM. KEY POINTS: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, the molecular determinants of hyperpolarization-activated current (I ) have been characterized as a 'pain pacemaker', and thus considered to be a potential molecular target for pain therapeutics. Dorsal root ganglion (DRG) neurons express Nav1.7, a channel that is not present in central neurons or cardiac tissue. Gain-of-function mutations (GOF) of Nav1.7 identified in inherited erythromelalgia (IEM), a human genetic model of neuropathic pain, produce DRG neuron hyperexcitability, which in turn produces severe pain. We found that I increases rheobase and reduces firing probability in small DRG neurons expressing WT Nav1.7, and demonstrate that the amplitude of subthreshold oscillations is reduced by I . We also demonstrate that I reverses the hyperexcitability of DRG neurons expressing a GOF Nav1.7 mutation (L858H) that causes IEM. Our results show that, in contrast to cardiac cells and CNS neurons, I acts to stabilize DRG neuron excitability and prevents excessive firing.
The link between sodium channel Nav1.7 and pain has been strengthened by identification of gain-of-function mutations in patients with inherited erythromelalgia (IEM), a genetic model of neuropathic pain in humans. A firm mechanistic link to nociceptor dysfunction has been precluded because assessments of the effect of the mutations on nociceptor function have thus far depended on electrophysiological recordings from dorsal root ganglia (DRG) neurons transfected with wild-type (WT) or mutant Nav1.7 channels, which do not permit accurate calibration of the level of Nav1.7 channel expression. Here, we report an analysis of the function of WT Nav1.7 and IEM L858H mutation within small DRG neurons using dynamic-clamp. We describe the functional relationship between current threshold for action potential generation and the level of WT Nav1.7 conductance in primary nociceptive neurons and demonstrate the basis for hyperexcitability at physiologically relevant levels of L858H channel conductance. We demonstrate that the L858H mutation, when modeled using dynamic-clamp at physiological levels within DRG neurons, produces a dramatically enhanced persistent current, resulting in 27-fold amplification of net sodium influx during subthreshold depolarizations and even greater amplification during interspike intervals, which provide a mechanistic basis for reduced current threshold and enhanced action potential firing probability. These results show, for the first time, a linear correlation between the level of Nav1.7 conductance and current threshold in DRG neurons. Our observations demonstrate changes in sodium influx that provide a mechanistic link between the altered biophysical properties of a mutant Nav1.7 channel and nociceptor hyperexcitability underlying the pain phenotype in IEM.
Ahn HS, Vasylyev DV, Estacion M, Macala LJ, Shah P , et al.
Brain research •
Sodium channel NaV1.7 is preferentially expressed in dorsal root ganglion (DRG) and sympathetic ganglion neurons. Gain-of-function NaV1.7 mutations/variants have been identified in the painful disorders inherited erythromelalgia and small-fiber neuropathy (SFN). DRG neurons transfected with these channel variants display depolarized resting potential, reduced current-threshold, increased firing-frequency and spontaneous firing. Whether the depolarizing shift in resting potential and enhanced spontaneous firing are due to persistent activity of variant channels, or to compensatory changes in other conductance(s) in response to expression of the variant channel, as shown in model systems, has not been studied. We examined the effect of wild-type NaV1.7 and a NaV1.7 mutant channel, D623N, associated with SFN, on resting potential and membrane potential during interspike intervals in DRG neurons. Resting potential in DRG neurons expressing D623N was depolarized compared to neurons expressing WT-NaV1.7. Exposure to TTX hyperpolarized resting potential by 7mV, increased current-threshold, decreased firing-frequency, and reduced NMDG-induced-hyperpolarization in DRG neurons expressing D623N. To assess the contribution of depolarized resting potential to DRG neuron excitability, we mimicked the mutant channel's depolarizing effect by current injection to produce equivalent depolarization; the depolarization decreased current threshold and increased firing-frequency. Voltage-clamp using ramp or repetitive action potentials as commands showed that D623N channels enhance the TTX-sensitive inward current, persistent at subthreshold membrane voltages, as predicted by a Hodgkin-Huxley model. Our results demonstrate that a variant of NaV1.7 associated with painful neuropathy depolarizes resting membrane potential and produces an enhanced inward current during interspike intervals, thereby contributing to DRG neuron hyperexcitability.