Limits...
Tuning Piezo ion channels to detect molecular-scale movements relevant for fine touch.

Poole K, Herget R, Lapatsina L, Ngo HD, Lewin GR - Nat Commun (2014)

Bottom Line: Piezo1 is the founding member of a class of mammalian stretch-activated ion channels, and we show that STOML3, but not other stomatin-domain proteins, brings the activation threshold for Piezo1 and Piezo2 currents down to ~10 nm.Structure-function experiments localize the Piezo modulatory activity of STOML3 to the stomatin domain, and higher-order scaffolds are a prerequisite for function.STOML3 is the first potent modulator of Piezo channels that tunes the sensitivity of mechanically gated channels to detect molecular-scale stimuli relevant for fine touch.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Max-Delbrück Center for Molecular Medicine, Robert-Rössle Straße 10, D-13092 Berlin, Germany.

ABSTRACT
In sensory neurons, mechanotransduction is sensitive, fast and requires mechanosensitive ion channels. Here we develop a new method to directly monitor mechanotransduction at defined regions of the cell-substrate interface. We show that molecular-scale (~13 nm) displacements are sufficient to gate mechanosensitive currents in mouse touch receptors. Using neurons from knockout mice, we show that displacement thresholds increase by one order of magnitude in the absence of stomatin-like protein 3 (STOML3). Piezo1 is the founding member of a class of mammalian stretch-activated ion channels, and we show that STOML3, but not other stomatin-domain proteins, brings the activation threshold for Piezo1 and Piezo2 currents down to ~10 nm. Structure-function experiments localize the Piezo modulatory activity of STOML3 to the stomatin domain, and higher-order scaffolds are a prerequisite for function. STOML3 is the first potent modulator of Piezo channels that tunes the sensitivity of mechanically gated channels to detect molecular-scale stimuli relevant for fine touch.

Show MeSH

Related in: MedlinePlus

STOML3 increasesPiezo1 and Piezo2 sensitivity.(a) Inverted epifluorescence images of N2a neuroblastoma cellsexpressing Lifeact-mCherry and STOML3-mGFP cultured on uncoated, PDMS pillar arrays(similar observations were made in 26 cells from 7 transfections (lifeAct)and in 19 cells from 10 transfections (STOML3-mGFP)). Inset is an overview of Lifeact-mCherrysignal in an individual cell; scale bars, 10 μm.(b) In individual cells, mechanically gated currents withvariable kinetics were observed: black traces N2a control cells; blue tracesN2a cells overexpressing STOML3-mGFP. (c–f)Stimulus–response data were binned and weighted by cell, anddisplayed as mean±s.e.m. and compared using Student’st-test where *P<0.05, **P<0.01,***P<0.001. (c) When endogenous Piezo1 was knocked down with miRNA(100 measurements, 10 cells) mechanosensitivity was significantly reducedcompared with control cells treated with scrambled miRNA (145 measurements,12 cells), Two-way analysis of variance (P<0.001), withBonferroni post-tests (**P<0.01). (d) Currentamplitudes were detected with stimuli much less than 100 nm inN2a cells overexpressing STOML3-mGFP (n=19 cells) compared with controlcells (n=26 cells), and knockdown of endogenous STOML3 messenger RNA led to astrong reduction in current amplitudes below control levels(**P<0.01; data compared with miRNA controls plotted in panelc). (e,f) Stimulus–response data ofmechanically gated currents in HEK-293 cells expressing Piezo1 (e) or Piezo2 (f) in the presenceor absence of STOML3. Asseen in N2a cells, the presence of STOML3 dramatically increased Piezo channel-mediatedmechanosensitivity in HEK-293 cells. (g) Co-immunoprecipitation ofPiezo1 withSTOML3 pulldown inHEK-293 cells. Experiment was repeated six times, and in each case bandscorresponding to Piezo proteins were detected in eluates from STOML3 pulldown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3973071&req=5

f5: STOML3 increasesPiezo1 and Piezo2 sensitivity.(a) Inverted epifluorescence images of N2a neuroblastoma cellsexpressing Lifeact-mCherry and STOML3-mGFP cultured on uncoated, PDMS pillar arrays(similar observations were made in 26 cells from 7 transfections (lifeAct)and in 19 cells from 10 transfections (STOML3-mGFP)). Inset is an overview of Lifeact-mCherrysignal in an individual cell; scale bars, 10 μm.(b) In individual cells, mechanically gated currents withvariable kinetics were observed: black traces N2a control cells; blue tracesN2a cells overexpressing STOML3-mGFP. (c–f)Stimulus–response data were binned and weighted by cell, anddisplayed as mean±s.e.m. and compared using Student’st-test where *P<0.05, **P<0.01,***P<0.001. (c) When endogenous Piezo1 was knocked down with miRNA(100 measurements, 10 cells) mechanosensitivity was significantly reducedcompared with control cells treated with scrambled miRNA (145 measurements,12 cells), Two-way analysis of variance (P<0.001), withBonferroni post-tests (**P<0.01). (d) Currentamplitudes were detected with stimuli much less than 100 nm inN2a cells overexpressing STOML3-mGFP (n=19 cells) compared with controlcells (n=26 cells), and knockdown of endogenous STOML3 messenger RNA led to astrong reduction in current amplitudes below control levels(**P<0.01; data compared with miRNA controls plotted in panelc). (e,f) Stimulus–response data ofmechanically gated currents in HEK-293 cells expressing Piezo1 (e) or Piezo2 (f) in the presenceor absence of STOML3. Asseen in N2a cells, the presence of STOML3 dramatically increased Piezo channel-mediatedmechanosensitivity in HEK-293 cells. (g) Co-immunoprecipitation ofPiezo1 withSTOML3 pulldown inHEK-293 cells. Experiment was repeated six times, and in each case bandscorresponding to Piezo proteins were detected in eluates from STOML3 pulldown.

Mentions: There has been debate over the molecular identity of stretch-activated channelsin mammalian cells18, but it is now clear that the Piezoproteins can form true mechanosensitive channels2526.Therefore, we next tested the effects of STOML3 on Piezo1-mediated currents in N2a neuroblastoma cells. N2acells were grown on uncoated pillar arrays and transfected with a plasmidencoding Lifeact-mCherry (Fig. 5a). In control N2a cells,pillar deflections evoked robust mechanosensitive currents with a surprisingdiversity of inactivation kinetics (Fig. 5b), includingrapidly inactivating currents and essentially non-inactivating currents (Fig. 5b; Supplementary Table 3); we could find no systematic relationshipbetween stimulus amplitude and inactivation kinetics (data not shown), but asimilar behaviour for Piezo1has been noted19. Compared with DRG neurons, relativelylarge pillar deflections were required to activate the N2a mechanosensitivecurrent (most often >200 nm) (Fig. 5c;see Supplementary Fig. 5 for allindividual data points). However, this stimulus size is considerably smallerthan that required for channel gating using cell indentation in the same celltype (>5 μm)26. Using microRNA(miRNA)-mediated reduction of Piezo1 expression, we could demonstrate an almost completeloss of mechanically gated currents observed within our stimulus range(0–1,000 nm) (Fig. 5c), clearlydemonstrating that the currents observed on pillar deflection are mediated bythe same channels measured with cell indentation.


Tuning Piezo ion channels to detect molecular-scale movements relevant for fine touch.

Poole K, Herget R, Lapatsina L, Ngo HD, Lewin GR - Nat Commun (2014)

STOML3 increasesPiezo1 and Piezo2 sensitivity.(a) Inverted epifluorescence images of N2a neuroblastoma cellsexpressing Lifeact-mCherry and STOML3-mGFP cultured on uncoated, PDMS pillar arrays(similar observations were made in 26 cells from 7 transfections (lifeAct)and in 19 cells from 10 transfections (STOML3-mGFP)). Inset is an overview of Lifeact-mCherrysignal in an individual cell; scale bars, 10 μm.(b) In individual cells, mechanically gated currents withvariable kinetics were observed: black traces N2a control cells; blue tracesN2a cells overexpressing STOML3-mGFP. (c–f)Stimulus–response data were binned and weighted by cell, anddisplayed as mean±s.e.m. and compared using Student’st-test where *P<0.05, **P<0.01,***P<0.001. (c) When endogenous Piezo1 was knocked down with miRNA(100 measurements, 10 cells) mechanosensitivity was significantly reducedcompared with control cells treated with scrambled miRNA (145 measurements,12 cells), Two-way analysis of variance (P<0.001), withBonferroni post-tests (**P<0.01). (d) Currentamplitudes were detected with stimuli much less than 100 nm inN2a cells overexpressing STOML3-mGFP (n=19 cells) compared with controlcells (n=26 cells), and knockdown of endogenous STOML3 messenger RNA led to astrong reduction in current amplitudes below control levels(**P<0.01; data compared with miRNA controls plotted in panelc). (e,f) Stimulus–response data ofmechanically gated currents in HEK-293 cells expressing Piezo1 (e) or Piezo2 (f) in the presenceor absence of STOML3. Asseen in N2a cells, the presence of STOML3 dramatically increased Piezo channel-mediatedmechanosensitivity in HEK-293 cells. (g) Co-immunoprecipitation ofPiezo1 withSTOML3 pulldown inHEK-293 cells. Experiment was repeated six times, and in each case bandscorresponding to Piezo proteins were detected in eluates from STOML3 pulldown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3973071&req=5

f5: STOML3 increasesPiezo1 and Piezo2 sensitivity.(a) Inverted epifluorescence images of N2a neuroblastoma cellsexpressing Lifeact-mCherry and STOML3-mGFP cultured on uncoated, PDMS pillar arrays(similar observations were made in 26 cells from 7 transfections (lifeAct)and in 19 cells from 10 transfections (STOML3-mGFP)). Inset is an overview of Lifeact-mCherrysignal in an individual cell; scale bars, 10 μm.(b) In individual cells, mechanically gated currents withvariable kinetics were observed: black traces N2a control cells; blue tracesN2a cells overexpressing STOML3-mGFP. (c–f)Stimulus–response data were binned and weighted by cell, anddisplayed as mean±s.e.m. and compared using Student’st-test where *P<0.05, **P<0.01,***P<0.001. (c) When endogenous Piezo1 was knocked down with miRNA(100 measurements, 10 cells) mechanosensitivity was significantly reducedcompared with control cells treated with scrambled miRNA (145 measurements,12 cells), Two-way analysis of variance (P<0.001), withBonferroni post-tests (**P<0.01). (d) Currentamplitudes were detected with stimuli much less than 100 nm inN2a cells overexpressing STOML3-mGFP (n=19 cells) compared with controlcells (n=26 cells), and knockdown of endogenous STOML3 messenger RNA led to astrong reduction in current amplitudes below control levels(**P<0.01; data compared with miRNA controls plotted in panelc). (e,f) Stimulus–response data ofmechanically gated currents in HEK-293 cells expressing Piezo1 (e) or Piezo2 (f) in the presenceor absence of STOML3. Asseen in N2a cells, the presence of STOML3 dramatically increased Piezo channel-mediatedmechanosensitivity in HEK-293 cells. (g) Co-immunoprecipitation ofPiezo1 withSTOML3 pulldown inHEK-293 cells. Experiment was repeated six times, and in each case bandscorresponding to Piezo proteins were detected in eluates from STOML3 pulldown.
Mentions: There has been debate over the molecular identity of stretch-activated channelsin mammalian cells18, but it is now clear that the Piezoproteins can form true mechanosensitive channels2526.Therefore, we next tested the effects of STOML3 on Piezo1-mediated currents in N2a neuroblastoma cells. N2acells were grown on uncoated pillar arrays and transfected with a plasmidencoding Lifeact-mCherry (Fig. 5a). In control N2a cells,pillar deflections evoked robust mechanosensitive currents with a surprisingdiversity of inactivation kinetics (Fig. 5b), includingrapidly inactivating currents and essentially non-inactivating currents (Fig. 5b; Supplementary Table 3); we could find no systematic relationshipbetween stimulus amplitude and inactivation kinetics (data not shown), but asimilar behaviour for Piezo1has been noted19. Compared with DRG neurons, relativelylarge pillar deflections were required to activate the N2a mechanosensitivecurrent (most often >200 nm) (Fig. 5c;see Supplementary Fig. 5 for allindividual data points). However, this stimulus size is considerably smallerthan that required for channel gating using cell indentation in the same celltype (>5 μm)26. Using microRNA(miRNA)-mediated reduction of Piezo1 expression, we could demonstrate an almost completeloss of mechanically gated currents observed within our stimulus range(0–1,000 nm) (Fig. 5c), clearlydemonstrating that the currents observed on pillar deflection are mediated bythe same channels measured with cell indentation.

Bottom Line: Piezo1 is the founding member of a class of mammalian stretch-activated ion channels, and we show that STOML3, but not other stomatin-domain proteins, brings the activation threshold for Piezo1 and Piezo2 currents down to ~10 nm.Structure-function experiments localize the Piezo modulatory activity of STOML3 to the stomatin domain, and higher-order scaffolds are a prerequisite for function.STOML3 is the first potent modulator of Piezo channels that tunes the sensitivity of mechanically gated channels to detect molecular-scale stimuli relevant for fine touch.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Max-Delbrück Center for Molecular Medicine, Robert-Rössle Straße 10, D-13092 Berlin, Germany.

ABSTRACT
In sensory neurons, mechanotransduction is sensitive, fast and requires mechanosensitive ion channels. Here we develop a new method to directly monitor mechanotransduction at defined regions of the cell-substrate interface. We show that molecular-scale (~13 nm) displacements are sufficient to gate mechanosensitive currents in mouse touch receptors. Using neurons from knockout mice, we show that displacement thresholds increase by one order of magnitude in the absence of stomatin-like protein 3 (STOML3). Piezo1 is the founding member of a class of mammalian stretch-activated ion channels, and we show that STOML3, but not other stomatin-domain proteins, brings the activation threshold for Piezo1 and Piezo2 currents down to ~10 nm. Structure-function experiments localize the Piezo modulatory activity of STOML3 to the stomatin domain, and higher-order scaffolds are a prerequisite for function. STOML3 is the first potent modulator of Piezo channels that tunes the sensitivity of mechanically gated channels to detect molecular-scale stimuli relevant for fine touch.

Show MeSH
Related in: MedlinePlus