Impact Of NaV1.7-PEPD Missense Mutations That Slow The Rate Of Inactivation On Sensory Neuronal Resurgent Sodium Currents

Abstract

Voltage-gated sodium (Nav1.1-9) channels are dynamic transmembrane proteins that, in response to changes in the potential across the lipophilic cell membrane, undergo specific conformational (gating) modifications, between ion-conducting (open) and non-conducting (closed and inactivated) states, to selectively conduct sodium ions through their aqueous pore. Importantly, changes in these voltage-dependent gating properties can impact action potential (AP) characteristics. TTX-sensitive sodium channels in cerebellar neurons can produce resurgent currents (Raman & Bean, 1997), intriguing currents that are re-activated during intermediate repolarizations following strong, but short, depolarizations. We observe resurgent currents in some DRG neurons and found that wild-type Nav1.6 but not wild-type Nav1.7 channels can generate resurgent currents in DRG neurons (Cummins et al., 2005). It has been demonstrated that, in cerebellar neurons from Nav1.6-null mice, slowing inactivation of the remaining Nav current can induce resurgent currents (Grieco & Raman, 2004). Interestingly, single-point missense mutations in the SCN9A gene that encode for Nav1.7, implicated in paroxysmal extreme pain disorder (PEPD), slow the rate of Nav1.7 inactivation (Jarecki et al., 2008). Therefore, we hypothesized that slowing of Nav1.7 by PEPD mutations might induce abnormal resurgent currents, thus altering AP properties. To explore this hypothesis, we transiently transfected adult rat DRG neurons with a TTX-resistant form of human Nav1.7-wild-type or PEPD mutant cDNA and rat Nav1.8-targeted shRNA. Voltage-dependent properties were observed using whole-cell voltage-clamp electrophysiology and AP generation was tested using current-clamp electrophysiology. Recordings were made in the presence and absence of extracellular TTX. These experiments should yield insight into (1) the mechanism of resurgent sodium current generation in DRG neurons, (2) a potential additive effect in channel dysfunction observed in PEPD, and (3) how these mutant channels contribute to alterations in AP characteristics.