NaV1.6 and NaV1.7 channels are major endogenous voltage-gated sodium channels in ND7/23 cells

Jisoo Lee,Hyun Jeong Kim,Frank H Yu,Hye-Mi Kim,Shinae Kim,Kevin P.M Currie

doi:10.1371/journal.pone.0221156

Abstract

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.

Highlights

Voltage-gated sodium channels (VGSC) are responsible for initiation and propagation of the action potential in central and peripheral neurons and most excitable cells such as heart and skeletal muscle [1]
By whole-cell patch clamp electrophysiology, robust inward peak sodium currents of –2.1 ±0.9nA (SE, Fig 1A) were recorded endogenously in these cells similar to prior studies [9, 18, 19] The activation and inactivation of the sodium currents were complete within ~2 milliseconds, consistent with the fast kinetics found in many TTX-sensitive (TTX-S) sodium channels
The aim of the present study was to determine the molecular profile of endogenous voltagegated sodium channels (VGSCs) that contribute to the sodium inward currents in ND7/23 cells

Summary

Introduction

Voltage-gated sodium channels (VGSC) are responsible for initiation and propagation of the action potential in central and peripheral neurons and most excitable cells such as heart and skeletal muscle [1]. The NaV1.8 and NaV1.9 channels show preferential expression in the peripheral sensory nerves, especially in the dorsal root ganglia (DRG) and trigeminal ganglia, and play critical roles in nociception and pain pathways [5]. These channels are important drug discovery targets for pain [6], yet our understanding of these channels have been limited because unlike many TTX-S sodium channels, recombinant NaV1.8 (TTX-R) is poorly expressed in many heterologous systems. Attempts to generate transient or stably-transfected HEK293 and CHO cells have been difficult [13], breakthrough stable expression of recombinant NaV1.9 in HEK293 was attained from a pharmaceutical industry research group recently [14]

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