Piezo2 is required for Merkel-cell mechanotransduction

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Abstract

How we sense touch remains fundamentally unknown1,2. The Merkel cell–neurite complex is a gentle touch receptor in the skin that mediates slowly adapting responses of Aβ sensory fibres to encode fine details of objects3,4,5,6. This mechanoreceptor complex was recognized to have an essential role in sensing gentle touch nearly 50 years ago3,4. However, whether Merkel cells or afferent fibres themselves sense mechanical force is still debated, and the molecular mechanism of mechanotransduction is unknown1,2,7,8,9,10,11,12. Synapse-like junctions are observed between Merkel cells and associated afferents6,13,14,15, and yet it is unclear whether Merkel cells are inherently mechanosensitive or whether they can rapidly transmit such information to the neighbouring nerve1,2,16,17. Here we show that Merkel cells produce touch-sensitive currents in vitro. Piezo2, a mechanically activated cation channel, is expressed in Merkel cells. We engineered mice deficient in Piezo2 in the skin, but not in sensory neurons, and show that Merkel-cell mechanosensitivity completely depends on Piezo2. In these mice, slowly adapting responses in vivo mediated by the Merkel cell–neurite complex show reduced static firing rates, and moreover, the mice display moderately decreased behavioural responses to gentle touch. Our results indicate that Piezo2 is the Merkel-cell mechanotransduction channel and provide the first line of evidence that Piezo channels have a physiological role in mechanosensation in mammals. Furthermore, our data present evidence for a two-receptor-site model, in which both Merkel cells and innervating afferents act together as mechanosensors. The two-receptor system could provide this mechanoreceptor complex with a tuning mechanism to achieve highly sophisticated responses to a given mechanical stimulus15,18,19.

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Figure 1: Piezo2 expression in hairy and glabrous skin.
Figure 2: Generation and characterization of skin-specific Piezo2 conditional knockout mice.
Figure 3: Mechanically activated currents in Merkel cells depend on Piezo2.
Figure 4: Ex vivo skin-saphenous nerve recordings in wild-type and Piezo2 conditional knockout mice.
Figure 5: Gentle touch assay in wild-type and Piezo2 conditional knockout mice.

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Acknowledgements

We would like to thank S. Murthy and B. Coste for suggestions. Research was supported by the Howard Hughes Medical Institute (to A.P.) and NIH grants R01DE022358 (to A.P.) and R01AR051219 (to E.A.L.).

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Contributions

S.-H.W. conducted experiments for Figs 13 and Extended Data Figs 15. A.E.D. conducted and analysed in vitro electrophysiology experiments in Fig. 3 and Extended Data Fig. 5. A.D.W. performed and analysed general skin–nerve recordings in Fig. 4a, b and Extended Data Figs 6 and 7. M.P. and K.R. performed behavioural experiments in Fig. 5. S.R. generated Piezo2fl/fl mice. Z.Q. and T.M. generated Piezo2GFP mice. During this manuscript’s peer-review process, we entered into a collaboration with Y.B. and E.A.L. E.A.L. conceived, and Y.B. performed and analysed targeted skin–nerve recordings in Fig. 4c–f. E.A.L. and C.L.S. contributed to the editing of the manuscript. S.-H.W. and A.P. designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Ardem Patapoutian.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Validation of anti-Piezo2 antibody.

a, Piezo2 detection by western blotting using anti-Piezo2 antibody (see full methods for antibody generation) in HEK293T cells overexpressing pIres2-EGFP (left lane), mPiezo1-pcDNA3.1(-)-Ires-EGFP (middle lane), and mPiezo2-sport6-Ires-EGFP (right lane). b, Piezo2 immunofluorescence in mPiezo2-sport6-Ires-EGFP-transfected HEK293T cells. The left panel shows EGFP epifluorescence in transfected cells, and the middle panel shows Piezo2 immunofluorescence in these same cells. c, d, Immunofluorescence of GFP and Piezo2 in adult Piezo2GFP reporter (c) and wild-type littermate (d) DRG. Piezo2 expression is observed in 45.6% of DRG neurons (587 neurons/1,287 total neurons). Of the Piezo2-expressing neurons, high Piezo2 expression was seen in 159/587 neurons. Scale bars (c, d), 100 μm.

Extended Data Figure 2 GFP immunofluorescence in wild-type control and Piezo2GFP reporter mice.

a, GFP, Krt8, and Nefh co-staining in wild-type littermate whisker follicle. b, c, GFP and Krt8 co-staining in wild-type littermate touch dome (b) and glabrous skin (c). Arrows mark the position of Krt8+ Merkel cells. dh, GFP and Nefh co-staining in Piezo2GFP whisker follicle. eh shows magnified views of the bracketed area in d. Arrows mark GFP expression only. Closed arrowheads mark the co-localization of GFP and Nefh. Scale bars (ah), 20 μm. epi, epidermis; der, dermis.

Extended Data Figure 3 Generation of Piezo2 null allele (Piezo2) and characterization of Piezo2 constitutive knockout mice.

a, A schematic diagram of Piezo2 allele generation. b, qRT–PCR (n = 2) showing Piezo2 levels in Piezo2wt/wt, Piezo2wt/−, and Piezo2−/− E19.5 lungs. Error bars represent mean ± s.e.m. **P < 0.01; NS, not significantly different, unpaired t-test with Welch’s correction. c, d, Piezo2 immunofluorescence in wild-type littermate (c) and Piezo2−/− newborn DRG (d). Scale bars (c, d), 100 μm.

Extended Data Figure 4 Characterization of Piezo2fl/fl (wild type) and Krt14Cre;Piezo2fl/fl (conditional knockout) adult skin.

a, b, Haematoxylin and eosin (H&E) staining of wild-type (WT) and Piezo2 conditional knockout (cKO) dorsal skin. c, d, Immunofluorescence of Krt14 and a-SMA (alpha smooth muscle actin) in wild-type and conditional knockout dorsal skin. eg, jl, Epidermal touch domes co-stained with Krt8, Nefh and VGLUT2 (vesicular glutamate transporter 2, a marker for Merkel cells) in wild-type and conditional knockout dorsal skin. h, m, n, Lanceolate endings and circumferential fibres co-stained with S100 (S100 calcium binding protein, a marker for Schwann cells) and Nefh in wild-type and conditional knockout dorsal skin. Closed arrowheads mark circumferential fibres, and arrows mark lanceolate endings. i, o, Meissner’s corpuscles co-stained with S100 and Nefh in wild-type and conditional knockout footpads. Closed arrows mark Meissner’s corpuscle. Scale bars, 100 μm (a, b), 20 μm (other panels). epi, epidermis; der, dermis.

Extended Data Figure 5 Current injections simulating Piezo2-mediated currents produce prolonged depolarizations in Merkel cells in vitro.

a, Representative traces of mechanically activated inward currents evoked by a gentle poking stimulus in a wild-type Merkel cell. A ramp (1 μm ms−1)-and-hold displacement stimulus (0.25 μm increments) was applied to the cell in whole-cell voltage clamp configuration. The steady state current at the end of a 125 ms displacement was −6 ± 2 pA (Vhold = −80 mV, n = 15), 4% of the maximal current observed (−146 ± 29 pA). b, Representative current clamp recordings from a wild-type Merkel cell, displaying a change in membrane potential in response to gentle mechanical stimuli. c, Piezo2-dependent currents were simulated by injecting short current pulses followed by different levels of long-lasting but small current injections. In wild-type Merkel cells (n = 4), membrane potential changes are elicited by applying a short (2.5 ms) 150 pA current injection followed by additional current injections of 0 pA (black), +0.5 pA (blue), +1 pA (orange) and +2 pA (red) from the bias holding current (−10 pA). In the absence of any continuous current, the membrane potential slowly decays after cessation of the initial 150 pA injection, consistent with a contribution of passive membrane properties (black trace). Importantly, long-lasting depolarizations are observed when these short pulses are followed by very small current injections (0.5–1 pA, which are 10–15% of the average observed Piezo2 current remaining at the end of the 125 ms mechanical stimulation (Fig. 3b, see above)). d, In wild-type cells (n = 4), membrane potential changes are elicited by a short 100 pA current injection followed by +3 pA (half the average Piezo2-dependent mechanically activated sustained current) from the bias holding current (−10 pA). Orange line, 2.5 ms initial pulse; Blue line, 5 ms initial pulse. These data indicate that long-lasting Piezo2 channel activity at levels below that observed during mechanical stimulation (panel a and Fig. 3b) is crucial for sustained membrane depolarizations in Merkel cells. The high Rm of these cells can enable small current fluctuations to produce large voltage fluctuations.

Extended Data Figure 6 Conduction velocity and von Frey thresholds of all slowly adapting Aβ afferents in wild-type and Piezo2 conditional knockout mice.

Conduction velocity (*P < 0.05, Student’s t-test) and von Frey thresholds (P = 0.0516, Mann–Whitney U-test) of all slowly adapting Aβ fibres from wild-type and Piezo2 conditional knockout mice. Error bars represent mean ± s.e.m.

Extended Data Figure 7 Characterization of rapidly adapting Aβ afferents in wild-type and Piezo2 conditional knockout mice.

a, Firing rates of rapidly adapting Aβ fibres in response to an increasing series of mechanical forces in wild-type and Piezo2 conditional knockout mice. b, c, Conduction velocity (b) and von Frey thresholds (c) of rapidly adapting Aβ fibres from wild-type and Piezo2 conditional knockout mice.

Extended Data Table 1 Membrane properties of wild-type and Piezo2 conditional knockout Merkel cells
Extended Data Table 2 Afferent properties of Aβ, Aδ, and C fibres from wild-type and Piezo2 conditional knockout animals
Extended Data Table 3 Summary of touch dome responses for wild-type and Piezo2 conditional knockout animals

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Woo, SH., Ranade, S., Weyer, A. et al. Piezo2 is required for Merkel-cell mechanotransduction. Nature 509, 622–626 (2014). https://doi.org/10.1038/nature13251

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