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Expression and Role of Voltage-Gated Sodium Channels in Human Dorsal Root Ganglion Neurons with Special Focus on Nav1.7, Species Differences, and Regulation by Paclitaxel.

ABSTRACT: Voltage-gated sodium channels (Navs) play an important role in human pain sensation. However, the expression and role of Nav subtypes in native human sensory neurons are unclear. To address this issue, we obtained human dorsal root ganglion (hDRG) tissues from healthy donors. PCR analysis of seven DRG-expressed Nav subtypes revealed that the hDRG has higher expression of Nav1.7 (~50% of total Nav expression) and lower expression of Nav1.8 (~12%), whereas the mouse DRG has higher expression of Nav1.8 (~45%) and lower expression of Nav1.7 (~18%). To mimic Nav regulation in chronic pain, we treated hDRG neurons in primary cultures with paclitaxel (0.1-1 μmol/L) for 24 h. Paclitaxel increased the Nav1.7 but not Nav1.8 expression and also increased the transient Na⁺ currents and action potential firing frequency in small-diameter (<50 μm) hDRG neurons. Thus, the hDRG provides a translational model in which to study "human pain in a dish" and test new pain therapeutics.

SUBMITTER: Chang W

PROVIDER: S-EPMC5648619 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Expression and Role of Voltage-Gated Sodium Channels in Human Dorsal Root Ganglion Neurons with Special Focus on Nav1.7, Species Differences, and Regulation by Paclitaxel.

Chang Wonseok W Berta Temugin T Kim Yong Ho YH Lee Sanghoon S Lee Seok-Yong SY Ji Ru-Rong RR

Neuroscience bulletin 20170419 1

Voltage-gated sodium channels (Navs) play an important role in human pain sensation. However, the expression and role of Nav subtypes in native human sensory neurons are unclear. To address this issue, we obtained human dorsal root ganglion (hDRG) tissues from healthy donors. PCR analysis of seven DRG-expressed Nav subtypes revealed that the hDRG has higher expression of Nav1.7 (~50% of total Nav expression) and lower expression of Nav1.8 (~12%), whereas the mouse DRG has higher expression of Na ...[more]

PMID: 28424991

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Project description:The molecular mechanisms of pain associated with alphaherpesvirus latency are not clear. We hypothesize that the voltage-gated sodium channels (VGSC) on the dorsal root ganglion (DRG) neurons controlling electrical impulses may have abnormal activity during latent viral infection and reactivation. We used herpes simplex virus 1 (HSV-1) to infect the human DRG-derived neuronal cell line HD10.6 in order to study the establishment and maintenance of viral latency, viral reactivation, and changes in the functional expression of VGSCs. Differentiated cells exhibited robust tetrodotoxin (TTX)-sensitive sodium currents, and acute infection significantly reduced the functional expression of VGSCs within 24 h and completely abolished VGSC activity within 3 days. A quiescent state of infection mimicking latency can be achieved in the presence of acyclovir (ACV) for 7 days followed by 5 days of ACV washout, and then the viruses can remain dormant for another 3 weeks. It was noted that during the establishment of HSV-1 latency, the loss of VGSC activity caused by HSV-1 infection could not be blocked by ACV treatment. However, neurons with continued ACV treatment for another 4 days showed a gradual recovery of VGSC functional expression. Furthermore, the latently infected neurons exhibited higher VGSC activity than controls. The overall regulation of VGSCs by HSV-1 during quiescent infection was proved by increased transcription and possible translation of Nav1.7. Together, these observations demonstrated a very complex pattern of electrophysiological changes during HSV infection of DRG neurons, which may have implications for understanding of the mechanisms of virus-mediated pain linked to latency and reactivation.IMPORTANCE The reactivation of herpesviruses, most commonly varicella-zoster virus (VZV) and pseudorabies virus (PRV), may cause cranial nerve disorder and unbearable pain. Clinical studies have also reported that HSV-1 causes postherpetic neuralgia and chronic occipital neuralgia in humans. The current work meticulously studies the functional expression profile changes of VGSCs during the processes of HSV-1 latency establishment and reactivation using human dorsal root ganglion-derived neuronal HD10.6 cells as an in vitro model. Our results indicated that VGSC activity was eliminated upon infection but steadily recovered during latency establishment and that latent neurons exhibited even higher VGSC activity. This finding advances our knowledge of how ganglion neurons generate uncharacteristic electrical impulses due to abnormal VGSC functional expression influenced by the latent virus.

Project description:ND7/23 cells are gaining traction as a host model to express peripheral sodium channels such as NaV1.8 and NaV1.9 that have been difficult to express in widely utilized heterologous cells, like CHO and HEK293. Use of ND7/23 as a model cell to characterize the properties of sodium channels requires clear understanding of the endogenous ion channels. To define the nature of the background sodium currents in ND7/23 cells, we aimed to comprehensively profile the voltage-gated sodium channel subunits by endpoint and quantitative reverse transcription-PCR and by whole-cell patch clamp electrophysiology. We found that untransfected ND7/23 cells express endogenous peak sodium currents that average -2.12nA (n = 15) and with kinetics typical of fast sodium currents having activation and inactivation completed within few milliseconds. Furthermore, sodium currents were reduced to virtually nil upon exposure to 100nM tetrodotoxin, indicating that ND7/23 cells have essentially null background for tetrodotoxin-resistant (TTX-R) currents. qRT-PCR profiling indicated a major expression of TTX-sensitive (TTX-S) NaV1.6 and NaV1.7 at similar levels and very low expression of TTX-R NaV1.9 transcripts. There was no expression of TTX-R NaV1.8 in ND7/23 cells. There was low expression of NaV1.1, NaV1.2, NaV1.3 and no expression of cardiac or skeletal muscle sodium channels. As for the sodium channel auxiliary subunits, β1 and β3 subunits were expressed, but not the β2 and β4 subunits that covalently associate with the α-subunits. In addition, our results also showed that only the mouse forms of NaV1.6, NaV1.7 and NaV1.9 sodium channels were expressed in ND7/23 cells that was originally generated as a hybridoma of rat embryonic DRG and mouse neuroblastoma cell-line. By molecular profiling of auxiliary β- and principal α-subunits of the voltage gated sodium channel complex, our results define the background sodium channels expressed in ND7/23 cells, and confirm their utility for detailed functional studies of emerging pain channelopathies ascribed to mutations of the TTX-R sodium channels of sensory neurons.

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Dataset Information

Expression and Role of Voltage-Gated Sodium Channels in Human Dorsal Root Ganglion Neurons with Special Focus on Nav1.7, Species Differences, and Regulation by Paclitaxel.

Publications

Expression and Role of Voltage-Gated Sodium Channels in Human Dorsal Root Ganglion Neurons with Special Focus on Nav1.7, Species Differences, and Regulation by Paclitaxel.

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