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

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.

Figure 1. Elastomeric pillar arrays for mechanical stimulation.

(a,b) SEM images of pillar arrays; scale bars 5 μm. (c) Acutely prepared DRG neuron expressing Lifeact-mGFP (green) cultured on EHS laminin/Cy3-coated pillar arrays (magenta); scale bar 20 μm. (d) Cultured DRG neuron neurites grow across the tops of pili (similar observations made in five cells, from three transfections); scale bar 5 μm. (e) Mechanical stimuli are applied to the neuron–laminin interface by applying a series of deflections to an individual pilus while recording from the cell with a whole-cell patch clamp. Black arrow indicates pilus being moved; attached neurite is outlined in yellow; scale bar 5 μm. (f) The centre point (red arrow) is determined using a 2D-Gaussian fit of intensity values; scale bar 1 μm. (g) Nanoscale stimuli applied at the neuron–substrate interface activate RA, IA or SA currents (observations made in 23 cells).

Figure 2. Comparing modes of mechanical stimulation.

(a) RA, IA and SA currents were observed on mechanical indentation of the soma, neurites or on deflection of a neurite-bound pilus. (b) On neurite indentation and pillar deflection, a higher variability was observed in the current types measured within a single cell (χ² test; ***P<0.001). Data collected from 23 cells in all three conditions; soma and neurite indentation represent matched data; pillar deflection is data from a separate set of cells. (c) Schematic representation of mechanical stimulation. Cell indentation studies require propagation of the physical stimulus via the cell to the cell-substrate interface. In contrast, deflecting a single pilus allows a defined stimulus to be applied directly at localized cell–matrix contacts.

Figure 3. Mechanically gated currents in mechanoreceptors and nociceptors.

(a) Mechanically gated currents in mechanoreceptors (narrow AP, no hump in the falling phase) were exclusively transient RA and IA currents; in nociceptors (broad APs, hump on falling phase) RA, IA and SA currents were observed. (b) A stimulus–response plot of individual data points (blue–mechanoreceptors, n=243 data points/17 cells; magenta–nociceptors, n=110 data points/13 cells) collected on array with k=290 pN m⁻¹ (insert: SEM image of pillar array, scale bar 5 μm). (c) The latency of channel gating was significantly shorter in mechanoreceptors (n=18) versus nociceptors (n=13, Student’s t-test, **P<0.01). (d,e) Mechanoreceptor APs can be classed into two additional subgroups, type I (blue) and type II (green), based on width (full-width at half-maximum) and duration of recovery after hyperpolarization. (f) Binned data indicate the higher sensitivity of type II compared with type I mechanoreceptors; current amplitudes were averaged for each bin and averages compared (type I: n=9 cells; type II: n=8 cells). To test for significance, Student's t-test was used; data are displayed as mean±s.e.m. (g) Representative currents from type I mechanoreceptors (blue trace; black line indicates τ₂ fit, 2 ms) and for type II mechanoreceptors (green traces; black line indicates τ₂ fit, 5.0 ms and 49). (h) Individual type I mechanoreceptors displayed either exclusively RA currents or exclusively IA currents. A mixture of both RA and IA currents was observed in individual type II mechanoreceptors. (i) Activation time constant (τ₁) of mechanotransduction currents (n=55 currents, 9 type I cells; 74 currents, 8 type II cells), presented as mean±s.e.m. Data obtained for cells cultured on arrays where k=290 pN nm⁻¹.

Figure 4. Mechanotransduction currents in DRG neurons from stoml3^−/− mice.

(a) Acutely isolated DRG neurons expressing STOML3-mGFP (green) cultured on a pillar array coated with EHS laminin/Cy3 (magenta); scale bar 20 μm. (b) STOML3-mGFP is targeted preferentially to contact points; the insert indicates line scan of intensity corresponding to the yellow line (similar observations made in eight cells, from three transfections, on eight pillar arrays); scale bar 20 μm. (c) In stoml3^−/− DRG neurons, we observed RA, IA and SA currents. (d–f) All data were binned into stimulus sizes and current amplitudes were averaged for each bin and compared between C57Bl/6 mice and stoml3^−/− mice. Data are displayed as mean±s.e.m. (d) Type I mechanoreceptors from C57Bl/6 mice (n=9 cells) versus stoml3^−/− mice (n=7 cells). (e) Type II mechanoreceptors isolated from C57Bl/6 mice (n=8 cells) versus stoml3^−/− mice (n=8 cells). (f) Nociceptors from C57Bl/6 mice (n=13 cells) versus stoml3^−/− mice (n=9 cells). To test for significance Student's t-test was used; *P<0.05, **P<0.01, ***P<0.001. Data were obtained for cells cultured on arrays where k=290 pN nm⁻¹.

Figure 5. STOML3 increases Piezo1 and Piezo2 sensitivity.

(a) Inverted epifluorescence images of N2a neuroblastoma cells expressing 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-mCherry signal in an individual cell; scale bars, 10 μm. (b) In individual cells, mechanically gated currents with variable kinetics were observed: black traces N2a control cells; blue traces N2a cells overexpressing STOML3-mGFP. (c–f) Stimulus–response data were binned and weighted by cell, and displayed as mean±s.e.m. and compared using Student’s t-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 reduced compared with control cells treated with scrambled miRNA (145 measurements, 12 cells), Two-way analysis of variance (P<0.001), with Bonferroni post-tests (**P<0.01). (d) Current amplitudes were detected with stimuli much less than 100 nm in N2a cells overexpressing STOML3-mGFP (n=19 cells) compared with control cells (n=26 cells), and knockdown of endogenous STOML3 messenger RNA led to a strong reduction in current amplitudes below control levels (**P<0.01; data compared with miRNA controls plotted in panel c). (e,f) Stimulus–response data of mechanically gated currents in HEK-293 cells expressing Piezo1 (e) or Piezo2 (f) in the presence or absence of STOML3. As seen in N2a cells, the presence of STOML3 dramatically increased Piezo channel-mediated mechanosensitivity in HEK-293 cells. (g) Co-immunoprecipitation of Piezo1 with STOML3 pulldown in HEK-293 cells. Experiment was repeated six times, and in each case bands corresponding to Piezo proteins were detected in eluates from STOML3 pulldown.

Figure 6. Desensitization of mechanosensitive currents in N2a cells is prevented by STOML3 and STOML1.

(a) Example traces from successive pillar deflections in control N2a cells (black traces), in N2a cells overexpressing STOML3-mGFP (cyan traces) or STOML1-mCherry (green traces). Stimuli were applied ~5 s apart. (b–e) Successive suprathreshold stimuli led to current desensitization in N2a cells (n=22 stimulation points). However, when either (b) STOML3 (n=25 stimulation points) or (c) STOML1 (n=19 stimulation points) is present, mechanosensitive currents do not desensitize as fast as controls; two-way analysis of variance (P<0.001). In addition, Bonferroni post-tests indicate that the probability of measuring a current in response to the second, third and fourth suprathreshold stimuli is significantly higher in those cells overexpressing STOML3 or overexpressing STOML1 (*P<0.05, **P<0.01, ***P<0.001). This desensitization is not modulated in the presence of (d) stomatin (n=15 stimulation points), (e) podocin (n=23 stimulation points) or (f) MEC-2 (n=13 stimulation points). Data were obtained for cells cultured on arrays where k=290 pN nm⁻¹. Control data are re-plotted in each panel for comparison, all data presented as mean±s.e.m.

Figure 7. Residues required for oligomerization and correct localization are necessary for STOML3 function.

(a) Stimulus–response curves for N2a cells overexpressing LifeAct-mCherry and fluorescently tagged STOML3 variants. Data were binned by stimulus magnitude and current amplitudes within each bin averaged for each cell and then averaged between cells, presented as mean±s.e.m; STOML3-V190P-mGFP (magenta squares; 172 measurements, 15 cells), STOML3-R90A-mGFP (grey squares; 218 measurements, 15 cells), STOML3-LR89,90EE-mGFP (green squares; 136 measurements, 13 cells), STOML3-P40S-mGFP (black squares; 142 measurements, 15 cells). Data from wtSTOML3 overexpression are re-plotted here for comparison (cyan squares) (b) Inverted epifluorescent images of STOML3 variants. Note that STOML3-V190P is localized similarly to wtSTOML3; STOML3-R90A and –LR89,90EE are localized in part to the membrane, but the vesicular fraction is lost and STOML3-P40S does not seem to be localized at the membrane nor in a vesicle pool (observations made on 15 cells/4 transfections). Scale bar 10 μm. (c) BiFC assays were used as a cell-based assay for oligomerization. In all cases wtSTOML3-VN was used as bait and as a control wtSTOML3-VC as prey. For experiments conducted on a single day, average slope of YFP signal development for the control was calculated and used to normalize all data. Data are displayed as mean±s.e.m. Oligomerization was significantly reduced when the STOML3-V190P-VC, STOML3-R90A-VC and LR89,90EE-VC variants were used as prey, in comparison with controls; Student’s t-test; ***P<0.001; n numbers indicate the number of transfections.

Figure 8. The stomatin domain of STOML3 is necessary to sensitize Piezo currents in N2a cells.

(a) Inverted epifluorescence images of Chimera1-mGFP and Chimera2-mCherry in N2a cells (observations made in 15 cells/4 transfections and 14 cells/4 transfections respectively; scale bar, 10 μm). Both chimeras localized to a vesicle pool and the plasma membrane. (b) Schematic representation of the two chimera proteins. (c) Stimulus–response data for Chimera1-mGFP (yellow circles; 220 measurements, 15 cells) and Chimera2-mCherry (blue circles; 299 measurements, 14 cells) in N2a cells. Control N2a cell data re-plotted here for comparison (open triangles) Data were binned by stimulus magnitude and current amplitudes within each bin averaged for each cell and then between cells, presented as mean±s.e.m. To test for significance, Student's t-test was used; *P<0.05. Chimera2, containing the stomatin-domain from STOML3, sensitized mechanically mediated currents more effectively than did Chimera1.