Endogenous Voltage-gated Sodium Channels of ND7/23 Cells

Objectives: Voltage-gated sodium channels (VGSC) underlie action potentials generated in excitable cells such as muscle and nerve. Tetrodotoxin (TTX; a toxin from puffer fish)-sensitive Na_V1.7, and TTX-resistant Na_V1.8 and Nav1.9 have essential and distinct roles in sensory transduction, such as in orofacial pain signaling. Recently, ND7/23, a fusion cell-line from rat DRG and mouse neuroblastoma, was used to study TTX-resistant sodium channels, despite having robust TTX-sensitive endogenous sodium currents. To better understand the nature of background sodium currents in ND7/23 cells, we profiled VGSC expression by molecular and electrophysiological methods.
Methods: Whole cell patch-clamp methods were used to characterize sodium currents in ND7/23 cells. For RT-PCR, cDNA was reverse transcribed from total RNA using oligo-dT or gene-specific primers. To distinguish whether transcript originated from mouse or rat, presence or absence of EcoR1 restriction site was used to analyze Na_V1.7 and Na_V1.8 amplicons by agarose gel size fractionation. To discriminate Na_V1.9, individual mouse- and rat-specific primer sets were used and amplicons then sequenced.
Results: Endogenous sodium currents were readily recorded in ND7/23 cells, with peak sodium currents that averaged –2.2±0.1 nA, and half-maximal activation at -22 mV and steady-state inactivation at -68 mV and TTX-sensitive inhibition (IC₅₀ ≈ 3.0 nM). Among the peripheral sodium channels, Na_V1.7 and Na_V1.9 transcripts were present in ND7/23 cells. Robust levels of Na_V1.6 were also expressed as well as minor levels of Na_V1.1, Na_V1.2, Na_V1.3 transcripts. Insensitivity to EcoR1 restriction suggested that Na_V1.7 amplicon was of mouse origin, and analysis by gene-specific primers indicated the absence of rat Na_V1.8 and Na_V1.9. Quantitative RT-PCR analysis showed that Na_V1.6 and Na_V1.7 transcripts were expressed at similar levels and Na_V1.9 transcripts were approximately 200-fold lower in ND7/23.
Conclusions: In ND7/23, only the mouse sodium channel genes were expressed. Absence of Na_V1.8 and minor expression Na_V1.9 transcripts correlated with inability to record endogenous TTX-resistant sodium currents in ND7/23 cells. TTX-sensitive endogenous sodium currents of ND7/23 cells were due to Na_V1.6 and Na_V1.7 transcripts that were approximately 200-fold greater than Na_V1.9.