Mechanosensitive ion Channels in Human Merkel Cells

Objectives: Merkel cells (MCs) participate in MC-neurite complexes with sensory neurons. We previously reported that MCs are capable of detecting cellular deformation via activation of transient receptor potential vanilloid (TRPV) subfamily members 1, 2, and 4, and the TRP ankyrin subfamily member-1 channel in hamsters. We have also reported that hamster MCs can be induced to release glutamate in response to cellular deformation resulting from direct mechanical stimulation. The released glutamate can activate NMDA receptors on rat trigeminal ganglion neurons. To date, no mechanoreceptive response in human MCs has been reported. We thus investigated the expression of functional mechanosensitive ion channels in human MCs.
Methods: We measured intracellular free Ca²⁺ concentration ([Ca²⁺]_i) using the fluorescent calcium indicator (fura-2). We applied direct mechanical stimulation using a glass micropipette. We used vectors encoding short hairpin RNA sequences specific for human Piezo1 to generate the Piezo1 gene silenced MCs, and an empty vector control. Non-parametric or parametric statistical significance were determined using Kruskal-Wallis or Friedman test with Dunn’s post-hoc test, or one-way ANOVA with Tukey’s post-hoc test. A P-value < 0.05 was considered significant.
Results: Immunofluorescence staining revealed that human MCs expressed cytokeratin 8, 14, 20, Piezo1, and Piezo2 channels. Yoda1 is a pharmacological agonist of the Piezo1 channel, and [Ca²⁺]_i transiently increased when Yoda1 was applied to MCs. This Yoda1 induced-[Ca²⁺]_i increase was significantly suppressed by application of Dooku1, a pharmacological antagonist of the Piezo1 channel. Transient increases in [Ca²⁺]_i induced by direct stimulation were not significantly inhibited by Dooku1. We observed no effects on [Ca²⁺]_i increases induced by direct mechanical stimulation in shRNA-Piezo1 transfected cells, compared to those observed in shRNA-Control transfected cells.
Conclusions: These results suggest that mechanosensitive ion channels other than the Piezo1 channel may specifically respond to mechanical stimulation in human MCs.