Characterization of Mechanosensitive Channels in Dental Primary Afferent System

Abstract

Dentin hypersensitivity is the acute, severe pain evoked by subtle stimulation on exposed dentin. This paradoxical pain has raised the need to identify the molecular and functional expression of mechanically sensitive ion channels in the dental pulp. Piezo ion channels are the mechanosensitive ion channels expressed in neurons and various cells of internal structure for sensation and regulation. Here I examined the molecular and functional expression of Piezo ion channels in dental primary afferent system by using RT-PCR, single-cell RT-PCR, in situ hybridization and whole cell patch clamp. Piezo1 mRNA was detected in whole trigeminal ganglion and pulp tissue by RT-PCR. However Piezo1 was not detected by single-cell RT-PCR in DiI-labeled dental primary afferent (DPA) neurons nor in odontoblasts. When odontoblasts and DPA neurons were examined for Piezo2, Piezo2 mRNA was not detected in odontoblasts, but were detected in DPA neurons. DPA neurons with Piezo2 mRNA or Piezo2-like currents were further characterized based on their neurochemical and electrophysiological properties. Piezo2 mRNA was found mostly in medium- to large-sized DPA neurons, with the majority of these neurons also positive for Nav1.8, CGRP and NF200, whereas only a minor population were positive for TRPV1, IB4 and peripherin. Whole cell patch clamp recordings revealed Piezo2-like, rapidly inactivating currents evoked by mechanical stimulation in a subpopulation of DPA neurons. Rapidly inactivating currents were pharmacologically blocked by ruthenium red, a compound known to block Piezo2 and were also reduced by siRNA-mediated Piezo2 knockdown. Piezo2-like currents were observed almost exclusively in IB4-negative DPA neurons, with the current amplitude larger in capsaicin-insensitive DPA neurons than the capsaicin-sensitive population. My findings show that subpopulation of DPA neurons are indeed mechanically sensitive. Within this subpopulation of mechanosensitive DPA neurons, I have identified the Piezo2 ion channel as a potential transducer for mechanical stimuli, contributing to rapidly inactivating inward currents. Piezo2-positive DPA neurons were characterized as medium to large-sized neurons with myelinated A-fibers, containing nociceptive peptidergic neurotransmitters.