The role of Drosophila Piezo in mechanical nociception

Abstract

Transduction of mechanical stimuli by receptor cells is essential for senses such as hearing, touch and pain. Ion channels have a role in neuronal mechanotransduction in invertebrates; however, functional conservation of these ion channels in mammalian mechanotransduction is not observed. For example, no mechanoreceptor potential C (NOMPC), a member of transient receptor potential (TRP) ion channel family, acts as a mechanotransducer in Drosophila melanogaster and Caenorhabditis elegans; however, it has no orthologues in mammals. Degenerin/epithelial sodium channel (DEG/ENaC) family members are mechanotransducers in C. elegans and potentially in D. melanogaster; however, a direct role of its mammalian homologues in sensing mechanical force has not been shown. Recently, Piezo1 (also known as Fam38a) and Piezo2 (also known as Fam38b) were identified as components of mechanically activated channels in mammals. The Piezo family are evolutionarily conserved transmembrane proteins. It is unknown whether they function in mechanical sensing in vivo and, if they do, which mechanosensory modalities they mediate. Here we study the physiological role of the single Piezo member in D. melanogaster (Dmpiezo; also known as CG8486). Dmpiezo expression in human cells induces mechanically activated currents, similar to its mammalian counterparts. Behavioural responses to noxious mechanical stimuli were severely reduced in Dmpiezo knockout larvae, whereas responses to another noxious stimulus or touch were not affected. Knocking down Dmpiezo in sensory neurons that mediate nociception and express the DEG/ENaC ion channel pickpocket (ppk) was sufficient to impair responses to noxious mechanical stimuli. Furthermore, expression of Dmpiezo in these same neurons rescued the phenotype of the constitutive Dmpiezo knockout larvae. Accordingly, electrophysiological recordings from ppk-positive neurons revealed a Dmpiezo-dependent, mechanically activated current. Finally, we found that Dmpiezo and ppk function in parallel pathways in ppk-positive cells, and that mechanical nociception is abolished in the absence of both channels. These data demonstrate the physiological relevance of the Piezo family in mechanotransduction in vivo, supporting a role of Piezo proteins in mechanosensory nociception.

Figure 1. Mechanical nociception defect in dpiezo knockout larvae

(a) Genomic map showing wild type dpiezo gene (upper) and engineered dpiezo KO (lower). Yellow and black boxes represent coding and non-coding exons, respectively. Deleted genomic segment in dpiezo KO is marked with a gray box. (b) Mechanical nociception assay using a range of stimulus forces in wild type and dpiezo KO larvae. n = 40 from four independent experiments. * p < 0.05, ** p < 0.01 from two tail paired Student t-test. (c, d) Mechanical nociception assay using repeated stimuli of the same larvae. n = 40. (e) Mechanical nociception assay using a 45 mN von Frey filament with wild type (+/+), heterozygous KO (+/−), heterozygous deficiency (Def/+), homozygous KO (−/−) and trans-heterozygous KO (Def/−). n > 85. *** p < 0.001. (f) Thermal nociception assay using heated probe (45 °C). n = 60. (g) Gentle touch assay. n > 150.

Figure 2. dpiezo functions in ppk-positive type II sensory neurons

(a) Double fluorescence labeling using ppk-EGFP (green) and dPiezoP-GAL4 that drives the expression of the nucleus targeted UAS-DsRed-NLS (red). A representative high-magnification image shows one ppk-positive neuron (arrow). All three ppk-positive cells in each hemisegment expressed dpiezo in all segments. (b) Mechanical nociception assay with dpiezo knockdown larvae in ppk-expressing cells by ppk-GAL4 and UAS-dpiezo-RNAi. n > 85, *** p < 0.001. (c) Mechanical nociception assay in rescued dpiezo KO. GFP-dPiezo was expressed in ppk-cells using ppk-GAL4 and UAS-GFP-dPiezo. n > 60. ** p < 0.01.

Figure 3. dpiezo mediates mechanically-activated currents in ppk-positive neurons

(a) Representative currents elicited by negative pipette pressure (0 to −60 mmHg, Δ 10 mmHg) in cell-attached configuration at −80mV in wild type (left) and dpiezo^−/− (right). (b) Average peak current-to pressure relationship of stretch activated currents in wild type (n = 12 cells) and dpiezo^−/− (n =13 cells). Data points are mean ± SEM. fitted with a Boltzmann equation. ** P < 0.01, *** P < 0.001, Mann-Whitney test.

Figure 4. dpiezo and ppk function in parallel pathways

(a) Mechanical nociception assay using a 45 mN von Frey filament with double null mutant of dpiezo and painless. Single knockout strains were used as controls and the wild type strain is w¹¹¹⁸. n > 60. (b) Mechanical nociception assay on heterozygous larvae for dpiezo and/or pain. n (heterozygote dpiezo KO) = 74 from three trials, n (heterozygote painless¹) = 169 from five trials, n (trans-heterozygote) = 166 from five trials. (c) Mechanical nociception assay with PPK and Piezo knockdown. ppk and/or dpiezo RNAi were driven by ppk-GAL4. n > 90. * p < 0.5, *** p < 0.001. n (wild type, w¹¹¹⁸) = 66, n (UAS-ShiDN/+) = 75 and n (ppk-GAL4/+; UAS-ShiDN/+) = 73 with three trials. (d) Gentle touch sensitivity assay with ppk and dpiezo knockdown. Wild type is w¹¹¹⁸. n > 90. (e) Thermal nociception assay using 45 °C probe with ppk and dpiezo knockdown. n > 75.