Dynamic-clamp analysis of wild-type human Na_v1.7 and erythromelalgia mutant channel L858H

Abstract

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.

NaV1.9: a sodium channel linked to human pain

“…Computer simulations show that, even at densities as low as 20% of the density estimated to be present in DRG neurons, Na V 1.9 conductance depolarizes the resting potential of the cell 17 . However, the effect of the Na V 1.9-induced depolarization is similar to that observed for mutant Na V 1.7 channels from patients with inherited erythromelalgia (which causes DRG neuron hyperexcitability); depolarization of the resting potential by such mutant Na V 1.7 channels only accounts for a portion of the reduction in action potential threshold, which also results from the increase in sodium conductance 30,31 . Consistent with this finding, one study used guanosine-5-O-thiotriphosphate (GTPγS; which potentiates Na V 1.9 activity) to directly show that upregulation of the Na V 1.9 current lowers the threshold of current that is required to generate action potentials 29 (FIG.…”

Human Nav1.8: enhanced persistent and ramp currents contribute to distinct firing properties of human DRG neurons

“…Furthermore, gain-of-function mutations of Na v 1.8 have been found in human subjects with painful neuropathies; relatively subtle gain-of-function changes in channel biophysics due to these mutations markedly alter the excitability of DRG neurons (Faber et al 2012;Han et al 2014;Huang et al 2013), underscoring the potential importance of even small differences in human Na v 1.8 channel properties, compared with those in rodents, for pain signaling. Na v 1.8 mutations linked to small-fiber neuropathy have been functionally profiled after expression in rodent DRG neurons (Faber et al 2012;Han et al 2014;Huang et al 2013).…”

“…Furthermore, gain-of-function mutations of Na v 1.8 have been found in human subjects with painful neuropathies; relatively subtle gain-of-function changes in channel biophysics due to these mutations markedly alter the excitability of DRG neurons (Faber et al 2012;Han et al 2014;Huang et al 2013), underscoring the potential importance of even small differences in human Na v 1.8 channel properties, compared with those in rodents, for pain signaling. Na v 1.8 mutations linked to small-fiber neuropathy have been functionally profiled after expression in rodent DRG neurons (Faber et al 2012;Han et al 2014;Huang et al 2013). Although rodent DRG neurons provide a tractable heterologous expression system for the functional assays that have been used to assess these mutations (Dib-Hajj et al 2009), notable differences between rodent and human DRG sodium channels have been reported, including, e.g., an ϳ10-mV difference in voltage dependence of human Na v 1.9 currents compared with rodent Na v 1.9 currents (DibHajj et al 1999).…”

Differential aging‐related changes in neurophysiology and gene expression in IB4‐positive and IB4‐negative nociceptive neurons

“…Window currents are produced by the overlap of activation and inactivation curves and, when generated around resting membrane potentials (RMPs), indicate that persistent noninactivating current will be generated at rest, where it can modulate excitability (Crill, 1996; Vasylyev, Han, Zhao, Dib‐Hajj, & Waxman, 2014). …”