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A role for Piezo2 in EPAC1-dependent mechanical allodynia.

Eijkelkamp N, Linley JE, Torres JM, Bee L, Dickenson AH, Gringhuis M, Minett MS, Hong GS, Lee E, Oh U, Ishikawa Y, Zwartkuis FJ, Cox JJ, Wood JN - Nat Commun (2013)

Bottom Line: Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton.Piezo2 knockdown also enhanced thresholds for light touch.Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres.

View Article: PubMed Central - PubMed

Affiliation: Molecular Nociception Group, Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, UK. N.Eijkelkamp@umcutrecht.nl

ABSTRACT
Aberrant mechanosensation has an important role in different pain states. Here we show that Epac1 (cyclic AMP sensor) potentiation of Piezo2-mediated mechanotransduction contributes to mechanical allodynia. Dorsal root ganglia Epac1 mRNA levels increase during neuropathic pain, and nerve damage-induced allodynia is reduced in Epac1-/- mice. The Epac-selective cAMP analogue 8-pCPT sensitizes mechanically evoked currents in sensory neurons. Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton. In vivo, 8-pCPT induces long-lasting allodynia that is prevented by the knockdown of Epac1 and attenuated by mouse Piezo2 knockdown. Piezo2 knockdown also enhanced thresholds for light touch. Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres. These data indicate that the Epac1-Piezo2 axis has a role in the development of mechanical allodynia during neuropathic pain.

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Activation of Epac1 but not Epac2 sensitizes mechanically evoked Piezo2 currents.HEK293a cells were transfected with constructs encoding Piezo2+ empty pcDNA3 (EV, +8-pCPT, n=21; +vehicle, n=38), and (a) Piezo2+Epac1-YFP (+8-pCPT, n=26; +vehicle, n=30), or (b) Piezo2+Epac2-YFP (+8-pCPT, n=17, +vehicle, n=23). 8-pCPT (specific Epac activator) or vehicle was added and cells were voltage clamped at −70 mV in whole-cell configuration. (a/b) Mechanically evoked currents were elicited by increasing displacement of the cell membrane in ~0.9 μm increments. (c) Peak current elicited by the largest mechanical stimulus before whole-cell configuration was lost. (d) Threshold of activation was determined as the mechanical stimulus that elicited a current >20 pA. (e) Exemplar traces of currents in response to increasing membrane deformation of HEK293a cells expressing Piezo2+EV, Piezo2+Epac1 and Piezo2+Epac1+8-pCPT. All data are expressed as mean±s.e.m. (a–b) Data were analysed using two-way analysis of variance followed by the Bonferroni post hoc test. *P<0.05, **P<0.01, ***P<0.001. In c and d ‘*’ indicates significant difference compared with all other groups analysed by one-way analysis of variance followed by the Bonferroni post hoc test.
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f4: Activation of Epac1 but not Epac2 sensitizes mechanically evoked Piezo2 currents.HEK293a cells were transfected with constructs encoding Piezo2+ empty pcDNA3 (EV, +8-pCPT, n=21; +vehicle, n=38), and (a) Piezo2+Epac1-YFP (+8-pCPT, n=26; +vehicle, n=30), or (b) Piezo2+Epac2-YFP (+8-pCPT, n=17, +vehicle, n=23). 8-pCPT (specific Epac activator) or vehicle was added and cells were voltage clamped at −70 mV in whole-cell configuration. (a/b) Mechanically evoked currents were elicited by increasing displacement of the cell membrane in ~0.9 μm increments. (c) Peak current elicited by the largest mechanical stimulus before whole-cell configuration was lost. (d) Threshold of activation was determined as the mechanical stimulus that elicited a current >20 pA. (e) Exemplar traces of currents in response to increasing membrane deformation of HEK293a cells expressing Piezo2+EV, Piezo2+Epac1 and Piezo2+Epac1+8-pCPT. All data are expressed as mean±s.e.m. (a–b) Data were analysed using two-way analysis of variance followed by the Bonferroni post hoc test. *P<0.05, **P<0.01, ***P<0.001. In c and d ‘*’ indicates significant difference compared with all other groups analysed by one-way analysis of variance followed by the Bonferroni post hoc test.

Mentions: As Epac signalling sensitizes mechanically evoked RA currents, we also tested effects on Piezo2-mediated mechanically evoked currents. We used HEK293a cells expressing Piezo2 and Epac1 or Epac2 (HEK293a cells express very low levels of Epac1/2) and mechanically distended these cells. Coexpression of Epac1 with Piezo2 in HEK293 cells did not change the stimulus–response curve compared with expression of Piezo2 alone (Fig. 4a). However, the addition of Epac-selective 8-pCPT to cells expressing Piezo2 and Epac1 strongly shifted the stimulus–response curve to the left. At a distension of ~8 μm, currents increased by ~2.5-fold (Fig. 4a). Coexpression of Epac2 with Piezo2 or application of 8-pCPT to cells expressing Piezo2+Epac2 or Piezo2 alone did not change the stimulus–response curve (Fig. 4b). In Epac1 expressing cells, 8-pCPT also increased the maximal mechanically evoked inward current before whole-cell configuration was lost because of the strength of the mechanical stimulus and reduced the threshold of activation, whereas Epac2 had no effect on either parameter (Fig. 4c). In cells expressing hPiezo1 and Epac1, 8-pCPT-induced activation of Epac1 also shifted the stimulus response of mechanically evoked Piezo1 currents to the left and decreased thresholds of activation, whereas no effect was seen when Epac2 was coexpressed with hPiezo1 (Supplementary Fig. S2). Overall these data indicate that Epac1 but not Epac2 activation results in sensitization of mechanically evoked Piezo-dependent currents. We further tested whether activation of other signalling molecules known to be involved in the development of mechanical hypersensitivity such as PKA, protein kinase C (PKC) and Ca2+ sensitize Piezo2 currents. Increasing cytosolic Ca2+ from 50 nM to 1 μM in Piezo2 expressing HEK293 cells resulted in sensitization of the mechanically evoked current (Supplementary Fig. S3a,b) and a reduction in threshold for channel activation (Supplementary Fig. S3c). Elevating cytosolic calcium also produced a marked slowing of adaptation to the static mechanical stimulus at cell displacements ≥4 μm (Supplementary Fig. 3d). In contrast, activation of PKC by preincubation with the phorbol derivative PMA had no effect on hPiezo2 channel activity or threshold (Supplementary Fig. S3e,f). Similarly, activation of PKA by preincubation with 6-Bnz-cAMP, a selective agonist of PKA, which does not activate Epac, had no significant effect on the stimulus–response curve (Supplementary Fig. S3g,h).


A role for Piezo2 in EPAC1-dependent mechanical allodynia.

Eijkelkamp N, Linley JE, Torres JM, Bee L, Dickenson AH, Gringhuis M, Minett MS, Hong GS, Lee E, Oh U, Ishikawa Y, Zwartkuis FJ, Cox JJ, Wood JN - Nat Commun (2013)

Activation of Epac1 but not Epac2 sensitizes mechanically evoked Piezo2 currents.HEK293a cells were transfected with constructs encoding Piezo2+ empty pcDNA3 (EV, +8-pCPT, n=21; +vehicle, n=38), and (a) Piezo2+Epac1-YFP (+8-pCPT, n=26; +vehicle, n=30), or (b) Piezo2+Epac2-YFP (+8-pCPT, n=17, +vehicle, n=23). 8-pCPT (specific Epac activator) or vehicle was added and cells were voltage clamped at −70 mV in whole-cell configuration. (a/b) Mechanically evoked currents were elicited by increasing displacement of the cell membrane in ~0.9 μm increments. (c) Peak current elicited by the largest mechanical stimulus before whole-cell configuration was lost. (d) Threshold of activation was determined as the mechanical stimulus that elicited a current >20 pA. (e) Exemplar traces of currents in response to increasing membrane deformation of HEK293a cells expressing Piezo2+EV, Piezo2+Epac1 and Piezo2+Epac1+8-pCPT. All data are expressed as mean±s.e.m. (a–b) Data were analysed using two-way analysis of variance followed by the Bonferroni post hoc test. *P<0.05, **P<0.01, ***P<0.001. In c and d ‘*’ indicates significant difference compared with all other groups analysed by one-way analysis of variance followed by the Bonferroni post hoc test.
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f4: Activation of Epac1 but not Epac2 sensitizes mechanically evoked Piezo2 currents.HEK293a cells were transfected with constructs encoding Piezo2+ empty pcDNA3 (EV, +8-pCPT, n=21; +vehicle, n=38), and (a) Piezo2+Epac1-YFP (+8-pCPT, n=26; +vehicle, n=30), or (b) Piezo2+Epac2-YFP (+8-pCPT, n=17, +vehicle, n=23). 8-pCPT (specific Epac activator) or vehicle was added and cells were voltage clamped at −70 mV in whole-cell configuration. (a/b) Mechanically evoked currents were elicited by increasing displacement of the cell membrane in ~0.9 μm increments. (c) Peak current elicited by the largest mechanical stimulus before whole-cell configuration was lost. (d) Threshold of activation was determined as the mechanical stimulus that elicited a current >20 pA. (e) Exemplar traces of currents in response to increasing membrane deformation of HEK293a cells expressing Piezo2+EV, Piezo2+Epac1 and Piezo2+Epac1+8-pCPT. All data are expressed as mean±s.e.m. (a–b) Data were analysed using two-way analysis of variance followed by the Bonferroni post hoc test. *P<0.05, **P<0.01, ***P<0.001. In c and d ‘*’ indicates significant difference compared with all other groups analysed by one-way analysis of variance followed by the Bonferroni post hoc test.
Mentions: As Epac signalling sensitizes mechanically evoked RA currents, we also tested effects on Piezo2-mediated mechanically evoked currents. We used HEK293a cells expressing Piezo2 and Epac1 or Epac2 (HEK293a cells express very low levels of Epac1/2) and mechanically distended these cells. Coexpression of Epac1 with Piezo2 in HEK293 cells did not change the stimulus–response curve compared with expression of Piezo2 alone (Fig. 4a). However, the addition of Epac-selective 8-pCPT to cells expressing Piezo2 and Epac1 strongly shifted the stimulus–response curve to the left. At a distension of ~8 μm, currents increased by ~2.5-fold (Fig. 4a). Coexpression of Epac2 with Piezo2 or application of 8-pCPT to cells expressing Piezo2+Epac2 or Piezo2 alone did not change the stimulus–response curve (Fig. 4b). In Epac1 expressing cells, 8-pCPT also increased the maximal mechanically evoked inward current before whole-cell configuration was lost because of the strength of the mechanical stimulus and reduced the threshold of activation, whereas Epac2 had no effect on either parameter (Fig. 4c). In cells expressing hPiezo1 and Epac1, 8-pCPT-induced activation of Epac1 also shifted the stimulus response of mechanically evoked Piezo1 currents to the left and decreased thresholds of activation, whereas no effect was seen when Epac2 was coexpressed with hPiezo1 (Supplementary Fig. S2). Overall these data indicate that Epac1 but not Epac2 activation results in sensitization of mechanically evoked Piezo-dependent currents. We further tested whether activation of other signalling molecules known to be involved in the development of mechanical hypersensitivity such as PKA, protein kinase C (PKC) and Ca2+ sensitize Piezo2 currents. Increasing cytosolic Ca2+ from 50 nM to 1 μM in Piezo2 expressing HEK293 cells resulted in sensitization of the mechanically evoked current (Supplementary Fig. S3a,b) and a reduction in threshold for channel activation (Supplementary Fig. S3c). Elevating cytosolic calcium also produced a marked slowing of adaptation to the static mechanical stimulus at cell displacements ≥4 μm (Supplementary Fig. 3d). In contrast, activation of PKC by preincubation with the phorbol derivative PMA had no effect on hPiezo2 channel activity or threshold (Supplementary Fig. S3e,f). Similarly, activation of PKA by preincubation with 6-Bnz-cAMP, a selective agonist of PKA, which does not activate Epac, had no significant effect on the stimulus–response curve (Supplementary Fig. S3g,h).

Bottom Line: Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton.Piezo2 knockdown also enhanced thresholds for light touch.Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres.

View Article: PubMed Central - PubMed

Affiliation: Molecular Nociception Group, Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, UK. N.Eijkelkamp@umcutrecht.nl

ABSTRACT
Aberrant mechanosensation has an important role in different pain states. Here we show that Epac1 (cyclic AMP sensor) potentiation of Piezo2-mediated mechanotransduction contributes to mechanical allodynia. Dorsal root ganglia Epac1 mRNA levels increase during neuropathic pain, and nerve damage-induced allodynia is reduced in Epac1-/- mice. The Epac-selective cAMP analogue 8-pCPT sensitizes mechanically evoked currents in sensory neurons. Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton. In vivo, 8-pCPT induces long-lasting allodynia that is prevented by the knockdown of Epac1 and attenuated by mouse Piezo2 knockdown. Piezo2 knockdown also enhanced thresholds for light touch. Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres. These data indicate that the Epac1-Piezo2 axis has a role in the development of mechanical allodynia during neuropathic pain.

Show MeSH
Related in: MedlinePlus