Heterologous expression and functional analysis of rat Na V1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7–23

Abstract

The voltage-gated sodium channel Na V1.8 (SNS, PN3) is thought to be a molecular correlate of the dorsal root ganglion (DRG) tetrodotoxin resistant (TTX-R) Na + current. TTX-R/Na V1.8 is an attractive therapeutic drug target for inflammatory and neuropathic pain on the basis of its specific distribution in sensory neurones and its modulation by inflammatory mediators. However, detailed analysis of recombinant Na V1.8 has been hampered by difficulties in stably expressing the functional protein in mammalian cells. Here, we show stable expression and functional analysis of rat Na V1.8 (rNa V1.8) in the rat DRG/mouse N18Tg2 neuroblastoma hybridoma cell line ND7–23. Rat Na V1.8 Na + currents were recorded (789±89 pA, n =62, over 20-cell passages) that qualitatively resembled DRG TTX-R in terms of gating kinetics and voltage-dependence of activation and inactivation. The local anaesthetic drug tetracaine produced tonic inhibition of rNa V1.8 (mean IC 50 value 12.5 μM) and in repeated gating paradigms (2–10 Hz) also showed frequency-dependent block. There was a correlation between the ability of several analogues of the anticonvulsant/analgesic compound lamotrigine to inhibit TTX-R and rNa V1.8 ( r =0.72, P <0.001). RT-PCR analysis of wild type ND7–23 cells revealed endogenous expression of the β1 and β3 accessory Na + channel subunits—the possibility that the presence of these subunits assists and stabilises expression of rNa V1.8 is discussed. We conclude that the neuroblastoma ND7–23 cell line is a suitable heterologous expression system for rNa V1.8 Na + channels in that it allows stable expression of a channel with biophysical properties that closely resemble the native TTX-R currents in DRG neurones. This reagent will prove useful in the search for pharmacological inhibitors of rNa V1.8 as novel analgesics.