Voltage-Gated Sodium Channels Regulating Action Potential Generation in Itch-, Nociceptive-, and Low-Threshold Mechanosensitive Cutaneous C-Fibers

Abstract

We evaluated the effect of voltage-gated sodium channel 1 (Na1) blockers in three nonoverlapping C-fiber subtypes in the mouse skin: chloroquine (CQ)-sensitive C-fibers with high mechanical thresholds- second, CQ-insensitive, capsaicin-sensitive C-fibers with high mechanical thresholds- and CQ and capsaicin-insensitive C-fibers with a very low mechanical threshold-C-LTMs. Na1-blocking drugs were applied to the nerve terminal receptive fields using an innervated isolated dorsal mouse skin-nerve preparation where the drugs are delivered into the skin intra-arterially. We combined these studies with an analysis of the mRNA expression of the -subunits of Na1 in individual dorsal root ganglia neurons labeled from the same region of the skin. Our results show that virtually all nociceptors and itch C-fibers expressed the tetrodotoxin (TTX)-resistant channels Na1.8 and Na1.9. However, TTX applied selectively into the skin abolished the action potential firing in response to mechanical stimulation in 75% of the itch C-fibers, 100% of the nociceptors, and 100% of C-LTMs. Na1.7 was the most commonly expressed TTX-sensitive Na1 in all three C-fiber subtypes innervating the dorsal skin. Selectively blocking Na1.7 abolished responses in about 40% of itch C-fibers, 65% of nociceptors, but only 20% of C-LTMs. Blocking Na1.8 alone had no affect on the firing sensitivity of the C-fibers. However, in itch and nociceptive C-fibers where the activation was not inhibited with a Na1.7 blocker, adding the Na1.8 blocker silenced action potential discharge.

Complementary roles of murine NaV1.7, NaV1.8 and NaV1.9 in acute itch signalling

“…The contribution of several Na V channels to acute itch signalling of different stimuli, shown here, supports the suggestion that a simultaneous inhibition of different Na V channels is required to reach full abolishment. This is in line with recent studies on ex vivo DRG-nerve-skin preparations reporting that tetrodotoxin suppressed firing in 75% of the fibers, Na V 1.7 selective blockage in 40% of itch C-fibers, but the combination of Na V 1.7 and Na V 1.8 inhibition resulted in full abolishment of action potential discharge 18 . Contrary, in human case studies a loss-of-function mutation of Na V 1.7 resulted in absence of itch perception 44 .…”

“…Despite the high expression of Na V 1.8 in sensory neurons activated by itch and pain 18 as well as the occurrence of chronic pruritus in patients with a gain-of-function mutation 27 , the role of Na V 1.8 in itch signalling remains poorly understood. Here, we show that Na V 1.8 modulates scratch behaviour induced, in particular, by the strong pruritogens 5-HT and histamine.…”

“…Surprisingly, deletion of Na V 1.8 resulted in a significantly reduced scratching induced by histamine while pharmacological inhibition of Na V 1.8 failed to decrease scratch activity. Jurcakova et al have recently shown that Na V 1.8 inhibition alone does not affect firing response of murine C-fibers upon chloroquine treatment, and dual inhibition of Na V 1.7 and 1.8 is needed to abolish this firing 18 . The sufficiency of sole Na V 1.8 inhibition to reduce acute itch signalling upon endothelin but not histamine, may indicate a different weight of Na V channel participation during acute itch signalling depending on the distinct stimulus.…”

“…Recordings were controlled by SiMon (V2.0, Academic Medical Center, University of Amsterdam, the Netherlands) and analysed offline by Scratch Analysis (V1.13, Academic Medical Center, University of Amsterdam, the Netherlands). Movements were classified as scratching based on their frequency (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), the amplitude of the signals (above 300 mV) and a minimum of 4 repetitions. For this methodical approach a positive prediction value of 95% was shown before 9 .…”

“…Primary sensory neurons express virtually all subunits 16,17 . However, transcription analysis using single neuron RT-PCR and RNA sequencing have revealed a vast abundance of Na V 1.7, 1.8 and 1.9 in the slow conducting unmyelinated sensory neurons 18,19 . Complementary physiological studies have shown that these channels modulate pain signalling [20][21][22] .…”