Navβ4 Regulates Fast Resurgent Sodium Currents and Excitability in Sensory Neurons

Abstract

BackgroundIncreased electrical activity in peripheral sensory neurons including dorsal root ganglia (DRG) and trigeminal ganglia neurons is an important mechanism underlying pain. Voltage gated sodium channels (VGSC) contribute to the excitability of sensory neurons and are essential for the upstroke of action potentials. A unique type of VGSC current, resurgent current (INaR), generates an inward current at repolarizing voltages through an alternate mechanism of inactivation referred to as open-channel block. INaRs are proposed to enable high frequency firing and increased INaRs in sensory neurons are associated with pain pathologies. While Nav1.6 has been identified as the main carrier of fast INaR, our understanding of the mechanisms that contribute to INaR generation is limited. Specifically, the open-channel blocker in sensory neurons has not been identified. Previous studies suggest Navβ4 subunit mediates INaR in central nervous system neurons. The goal of this study was to determine whether Navβ4 regulates INaR in DRG sensory neurons.ResultsOur immunocytochemistry studies show that Navβ4 expression is highly correlated with Nav1.6 expression predominantly in medium-large diameter rat DRG neurons. Navβ4 knockdown decreased endogenous fast INaR in medium-large diameter neurons as measured with whole-cell voltage clamp. Using a reduced expression system in DRG neurons, we isolated recombinant human Nav1.6 sodium currents in rat DRG neurons and found that overexpression of Navβ4 enhanced Nav1.6 INaR generation. By contrast neither overexpression of Navβ2 nor overexpression of a Navβ4-mutant, predicted to be an inactive form of Navβ4, enhanced Nav1.6 INaR generation. DRG neurons transfected with wild-type Navβ4 exhibited increased excitability with increases in both spontaneous activity and evoked activity. Thus, Navβ4 overexpression enhanced INaR and excitability, whereas knockdown or expression of mutant Navβ4 decreased INaR generation.ConclusionINaRs are associated with inherited and acquired pain disorders. However, our ability to selectively target and study this current has been hindered due to limited understanding of how it is generated in sensory neurons. This study identified Navβ4 as an important regulator of INaR and excitability in sensory neurons. As such, Navβ4 is a potential target for the manipulation of pain sensations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12990-015-0063-9) contains supplementary material, which is available to authorized users.

Upregulation of the sodium channel NaVβ4 subunit and its contributions to mechanical hypersensitivity and neuronal hyperexcitability in a rat model of radicular pain induced by local dorsal root ganglion inflammation

“…Our results confirm and extend previous work showing that Na V β4 is primarily expressed in larger sensory neurons and that it plays a key role in mediating resurgent currents in inflamed, hypersensitive sensory neurons [4; 26; 53]. …”

FHF2 isoforms differentially regulate Nav1.6-mediated resurgent sodium currents in dorsal root ganglion neurons

“…Additionally, we knocked down endogenous TTXR Nav1.8 channels with a small hairpin RNA (shRNA) plasmid to aid in the isolation of Nav1.6r currents. The Nav1.8 shRNA-IRES-dsRED plasmid encoded for Nav1.8 shRNA sequence (targeting sequence, GATGAGGTCGCTGCTAAG, (37) and an internal ribosome entry site for the translation of fluorescent protein marker dsRed (IRES-dsRED) as previously described (3, 28). …”

“…For peptide studies, only Nav1.6r and Nav1.8 shRNA-IRES-dsRED were co-transfected. Expression of the Nav1.6r construct with Nav1.8 shRNA allowed us to study the modulation of Nav1.6r by auxiliary subunits in isolation from endogenous channels as previously described (3, 28). Although endogenous TTXR Nav1.8 channels run down in culture (17, 28), by using the Nav1.8 shRNA-IRES-dsRED plasmid we further decreased Nav1.8 to minimize contamination.…”

“…Sodium channel auxiliary subunits are potential candidates for resurgent current modulation. For example, we recently reported that sodium channel beta 4 subunit (Navβ4) is a major determinant of resurgent currents in DRG neurons (3). Our results were consistent with Central Nervous System (CNS) studies, which proposed the C-terminal sequence of this subunit acts as an open channel blocker (2, 21, 39).…”

Ion channel regulation by β-secretase BACE1 – enzymatic and non-enzymatic effects beyond Alzheimer's disease

“…63 Mechanistically, they result from transient channel re-openings upon repolarization, when an unorthodox open-channel block, most likely exerted by parts of the b4 subunit, is relieved. 64,65 Since BACE1, which is known to cleave b4, is highly expressed in cerebellum, an obvious question was whether Purkinje cells from BACE1-deficient mice would exhibit altered firing pattern. 66 When compared to wild type neurons, the spontaneous firing of Purkinje cells from mutant mice was indeed reduced (Fig.…”