ARTICLE
doi: 10.1038/nature 10812
Piezo proteins are pore-forming subunits of mechanically activated channels
Bertrand Coste1*, Bailong Xiao1*, Jose S. Santos2, Ruhma Syeda2, Jorg Grandly, Kathryn S. Spencer1, Sung Eun Kim1, Manuela Schmidt1, Jayanti Mathur3, Adrienne E. Dubin1, Mauricio Montal2 & Ardem Patapoutian1'3
Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezol (MmPiezol) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezol assembles as a ~1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezol reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction.
Mechanically activated currents have been described in various mammalian cells, including inner ear hair cells1, somatosensory dorsal root ganglion neurons2, vascular smooth muscle cells3 and kidney primary epithelia4. Most of these mechanically activated currents are cationic with Ca2+ permeability, leading to a search for cation channels able to convert mechanical forces into such currents. Few mechanically activated channels have been described so far"; however, none of the candidates has been shown convincingly to mediate the physiologically relevant non-selective cationic mechanically activated currents in mammals.
MmPiezol was recently identified as a protein required for mechanically activated currents in a mammalian cell line. Expressing MmPiezol or related MmPiezo2 in a variety of mammalian cell lines induces mechanically activated cationic currents8. MmPiezol -induced currents are inhibited by GsMTx4 (Grammostola spatulata mechanotoxin 4), a peptide widely used to study mechanically activated channels9. MmPiezol and MmPiezo2 contain over 30 putative transmembrane domains and do not resemble known ion channels or other protein classes. Piezo proteins could be non-conducting subunits of cationic ion channels required for proper expression or for modulating channel properties61011. Alternatively, Piezo proteins may define a novel class of ion channels involved in mechanotransduction.
Mechanosensitivity of DmPiezo
Piezo sequences are present in the genomes of many animal, plant and other eukaryotic species. Functional analysis of Piezo proteins from phylogenetically distant species could demonstrate a conserved role of these proteins in mechanotransduction; furthermore, a comparative analysis of mechanically activated currents could elucidate unique pore properties of channels induced by Piezo proteins from distinct species. We focused on the apparently single member of D. melanogaster Piezo (DmPiezo), as this invertebrate species is widely used to study mechanotransduction using genetic approaches1216. DmPiezo is 24% identical to mammalian Piezo proteins, with sequence conservation
throughout the length of the proteins (Supplementary Fig. 1). We cloned the full-length DmPiezo complementary DNA into pIRES2-EGFP vector. We recorded mechanically activated currents from fluorescent HEK293T cells expressing DmPiezo-pIRES2-EGFP by applying force to the cell surface while monitoring transmembrane currents at constant voltage using patch-clamp recordings in the whole-cell configuration21718. DmPiezo, but not mock-transfected cells, showed large mechanically activated currents (Fig. la, b). These currents have a time constant of inactivation t of 6.2 ± 0.3 ms (n = 32 cells) at — 80 mV when fitted with mono-exponential function, which is faster than observed for MmPiezol (—16 ms) and more comparable to MmPiezo2 (~7ms)8. Similar to its mammalian counterparts, DmPiezo mechanically activated currents are characterized by a linear current-voltage (I-V) relationship with a reversal potential around OmV, consistent with it mediating a non-selective cationic conductance (Fig. lc). We further characterized DmPiezo-induced currents in HEK293T cells in response to negative pressure pulses applied through the recording pipette in the cell-attached mode, an alternative mechanosensitivity assay. Overexpression of DmPiezo induced stretch-activated currents (Fig. Id, e) with a pressure for half-maximal activation (P50) of —31.8 ± 2.8mmHg (Fig. If), similar to the P50 calculated for MmPiezol-induced currents (—30 mmHg)8. Therefore, mechanosensitivity of the Piezo family is conserved in invertebrates. We demonstrate the physiological relevance of DmPiezo in vivo in an accompanying paper19.
Pore properties of Piezo proteins
We next compared fundamental permeation properties of MmPiezol and DmPiezo. Ruthenium red, a polycationic pore blocker of TRP channels20-21, blocks MmPiezol- and MmPiezo2-induced mechanically activated currents8. We found that ruthenium red is a voltage-dependent blocker of MmPiezol, with an IC50 value of 5.4 ± 0.9 uM at -80 mV (Fig. 2a-c): at a concentration of 30 uM, extracellular ruthenium red inhibited inward mechanically activated currents without affecting
department of Cell Biology, Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, California 92037, USA. 2Section of Neurobiology, Division of Biological Sciences, University of California San Diego, La Jolla, California 92093, USA. 3Genomic Institute of the Novartis Research Foundation, San Diego, California 92121, USA. fPresent address: Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA. These authors contributed equally to this work.
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Time (ms]
Figure 1 Human cells expressing Drosophila Piezo (DmPiezo) show large mechanically activated currents, a-f, Mechanically activated currents of DmPiezo-expressing HE293T cells recorded in the whole-cell (a-c) or cell-attached (d-f) configuration, a, Representative traces of mechanically activated inward currents at — 80 mV in DmPiezo-transfected cells subjected to a series of mechanical steps in 1 urn increments, b, Average maximal current amplitude of mechanically activated inward currents at — 80 mV. c, Representative I-V relationship of mechanically activated currents in DmPiezo-transfected cells. The inset shows mechanically activated currents evoked at holding potentials ranging from — 80 to +80 mV. d, Representative currents elicited by negative pipette pressure (0 to —60 mm Hg, A20 mm Hg) in DmPiezo-transfected cells.
e, Average maximal current amplitude of stretch-activated currents at — 80 mV.
f, 7max normalized current-pressure relationship of stretch-activated currents recorded at — 80 mV in DmPiezo-transfected cells (n = 8 cells) and fitted with a Boltzmann equation. P50 is the average of P50 values determined for individual cells. Bars represent mean ± s.e.m. and the number of cells tested is shown above bars. ***p< 0.001, Mann-Whitney [/-test.
outwards currents. Such voltage dependence is a characteristic of open channel block. A high concentration of ruthenium red (50 uM) included in the pipette solution in the whole-cell configuration showed no evidence of block, as large mechanically activated currents still displayed a linear I-V relationship (Supplementary Fig. 2). These results suggest that ruthenium red blocks the pore of MmPiezol -induced mechanically activated channels from the extracellular side. Notably, DmPiezo-induced mechanically activated currents were insensitive to ruthenium red concentrations that potently blocked MmPiezol-induced currents (Fig. 2d, e). Together, these results demonstrate that overexpression of DmPiezo or MmPiezol gives rise to mechanically activated channels with distinct channel properties.
Next, we set out to determine the single channel conductance (y) of mechanically activated channels induced by Piezo proteins by using negative-pressure stimulations of membrane patches in cell-attached mode. Figure 3 shows the single mechanically activated channel properties of MmPiezol or DmPiezo. Openings of stretch-activated channels showed a marked difference in amplitude of single channel currents (Fig. 3a), as determined from the single channel 7-V relationship for MmPiezol and DmPiezo (Fig. 3b, c). Linear regression of these I- V relationships resulted in slope-conductance values in these recording conditions of 29.9 ± 1.9 and 3.3 ± 0.3 pS for MmPiezol- and DmPiezo-induced mechanically activated currents, respectively (n = 7 and 5 cells; mean ± s.e.m.). Therefore, DmPiezo-dependent channels are ninefold less conductive than MmPiezol-dependent channels.
MmPiezol oligomerization
The pore of most ion channels is formed by the assembly of transmembrane domains from distinct subunits (for example, voltage-gated K+ channels, ligand-gated ion channels) or structurally repetitive domains within a large protein (for example, voltage gated Na+ and
Figure 2 Ruthenium red is a channel pore blocker of MmPiezol- but not DmPiezo-induced currents, a, Representative traces of mechanically activated currents in MmPiezol-transfected cells evoked at holding potentials ranging from — 80 to +80 mV before (left panel) and during perfusion of 30 uM of ruthenium red (right panel, red traces), b, Average I-V relationship of mechanically activated currents in MmPiezol-transfected cells (n = 7 cells) before (black symbols) and during (red symbols) perfusion of 30 uM ruthenium red. Currents were normalized to the value of control current evoked at — 80 mV for each individual cell, c, Concentration-inhibition curve for ruthenium red (RR) on mechanically activated currents evoked at — 80 mV in MmPiezol -transfected cells and fitted with a Boltzmann equation. Each data point is the mean ± s.e.m. of 3-13 observations, d, Representative traces of Piezo-dependent mechanically activated currents evoked at — 80 mV in the presence of ruthenium red. e, Blocking effect of ruthenium red on Piezo-dependent mechanically activated currents evoked at — 80 mV. Bars represent mean ± s.e.m. and the number of cells tested is shown above the bars. **P< 0.01; ***P < 0.001; unpaired f-test.
Ca2+ channels). As Piezo proteins lack repetitive transmembrane motifs presumably they oligomerize to form ion channels. To test this hypothesis, we determined the number of subunits in Piezo complexes by expressing GFP-MmPiezol fusion proteins in Xenopus laevis oocytes, imaging individual spots with total internal reflection microscopy (TIRF), and counting discrete photobleaching steps (Fig. 4a, b and ref. 22). Amino-terminal GFP-MmPiezol functionality was confirmed by overexpression in HEK293T cells (Supplementary Fig. 3). We used several GFP fusion constructs of ion channels with known stoichiometry as controls: voltage-gated Ca2+ channel (alE-GFP; monomer), NMDA (N-methyl-D-aspartate) receptor (NR1 co-expressed with NR3A-GFP; dimer of dimers) and cyclic nucleotide gated (CNG) channel (XfA4-GFP; tetramer)22. We found that complexes of MmPiezo 1 frequently exhibited at most four photobleaching steps, consistent with the idea that Piezo proteins homo-multimerize. Fluorescent MmPiezo 1 (or CNG) complexes exhibiting bleaching in fewer than four steps can be explained by non-functional GFP or pre-bleached GFP22 or general bias against noisier multi-step traces during data analysis (see Methods). Histograms of the number of photobleaching steps observed for MmPiezol complexes were comparable to histograms obtained from tetrameric CNG channels (Fig. 4c). These results suggest that in living cells, Piezo proteins can assemble as homo-multimers.
We further characterized Piezo proteins biochemically by heterolo-gously expressing and purifying MmPiezol carboxy-terminally fused with a glutathione S-transferase (MmPiezol-GST). Functionality of MmPiezol-GST was confirmed by overexpression in HEK293T cells (Supplementary Fig. 3). We observed a protein band at a position near the 260-kDa protein marker on a Coomassie-blue-stained denaturing protein gel (Supplementary Fig. 4a). Western blot with a GST {Schistosoma japonicum form) antibody (Supplementary Fig. 4b) or
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MmPiezol
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100 ms   I 5 pA 100 ms   I 1 pA
Average current _ Average current
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\/h = -180mV   \/h = -160mV \/h = -140mV   \/h = -120mV
ILiLlILILl
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(PA)
I 0.5 pA
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180-140-100 -60 -20
Figure 3 MmPiezol- and DmPiezo-induced stretch-activated channels have different conductances, a, Representative Piezo-dependent stretch-activated channel openings elicited at — 180 mV. Bottom traces represent average of 40 individual recording traces, b, All-point histograms of single channel opening events (average of 10 and 20 individual events for MmPiezol and DmPiezo, respectively) at different holding potentials (Vh). c, Average I-V relationships of stretch activated single channels in MmPiezol and DmPiezo transfected cells (n = 7 and 5 cells, respectively; mean ± s.e.m.). Single channel amplitude was determined as the amplitude difference in Gaussian fits as shown in b.
a MmPiezol-specific antibody8 (Fig. 4) confirmed the presence of MmPiezol-GST in the MmPiezol-GST sample. Using native gel electrophoresis and Coomassie blue staining, we detected a prominent protein band at a position near the 1,236 kDa protein marker only in the MmPiezol-GST sample (Fig. 4d). Western blot using MmPiezol antibody confirmed that this major band contains MmPiezol (Fig. 4e). These data indicate that the purified MmPiezol-GST protein complex has a molecular weight of about 1.2 million Da, four times the predicted molecular weight of a single MmPiezol-GST polypeptide (318 kDa). Next, we asked whether any endogenous proteins are present in this MmPiezol-containing complex. Mass spectrometry of the —1.2 million Da protein complex mainly detected peptides derived from MmPiezol-GST, but not from other endogenous membrane proteins. Although several non-transmembrane proteins were also detected, most of them were also present in the control sample, indicating an absence of specific interacting proteins in the complex (Supplementary Table 1). Moreover, mass spectrometry of the whole purified solution samples before gel electrophoresis confirmed that no other ion channel protein was detected (Supplementary Table 2). This indicates that MmPiezol is not tightly associated with any endogenous pore-forming protein.
To examine further whether this Piezo complex is indeed a tetramer, we treated the purified MmPiezol-GST protein with the crosslinker formaldehyde and subjected the samples to denaturing gel electrophoresis and western blotting. Formaldehyde-treated samples contained three major additional higher-order Piezo-containing bands, with longer treatments increasing the prominence of the higher bands (Fig. 4f). The distribution of the bands on the 3-8% gradient gel suggests that the four bands correspond to monomer, dimer, trimer and tetramer of MmPiezol-GST (Fig. 4f). The observation that MmPiezol is crosslinked by formaldehyde, a crosslinker with a relative short spacer arm (2.3-2.7 A), suggests that the subunits form a tetramer.
It is possible that MmPiezo 1 oligomers associate with other proteins; however, such an association might not withstand the GST purification step. To probe this, we performed paraformaldehyde (PFA) crosslink-ing experiments on living cells before the purification procedure. On a native gel, the MmPiezol-GST complex purified from PFA-treated cells also migrated to the position near the 1,236 kDa protein marker, similar to the sample from untreated cells (Fig. 4g). On a denaturing gel, on-cell PFA treatment resulted in four distinct MmPiezol-specific bands, similar to results of formaldehyde treatment on the purified complex (Fig. 4h). This suggests that MmPiezol is not tightly associated with other proteins large enough to alter discernibly its size on denaturing gels, and confirms the results from mass spectrometry analysis. However, our crosslinking studies with PFA might miss weak interactors with MmPiezol. Regardless, together with the results obtained from single-molecule photobleaching analysis in living cells, our biochemical data suggest that MmPiezol forms a homomultimeric ion channel, most likely as a homotetramer.
MmPiezol reconstitution in lipid bilayers
Finally, to assess whether Piezo proteins were sufficient to recapitulate the channel properties recorded from Piezo-overexpressing cells, we reconstituted purified MmPiezol proteins into lipid bilayers in two distinct configurations: droplet interface lipid bilayers (DIBs) assembled from two monolayers23-25 (Fig. 5a-e and 1-q) and proteoliposomes26 (Fig. 5f-h). In the first configuration, MmPiezol was reconstituted into asymmetric bilayers that mimic the cellular environment: the extracellular-facing lipid monolayer is predominantly neutral whereas the intracellular-facing leaflet is negatively charged27. In contrast, the lipid composition of the bilayer in the second configuration is uniform.
In the DIBs setting, representative segments from a 6-min recording obtained at —100 mV show brief, discrete channel openings (Fig. 5a, b) blocked by addition of 50 uM ruthenium red to the neutral facing compartment (Fig. 5c). In contrast, no effect was observed when ruthenium red was introduced into the negative-facing compartment (not shown). We detected efficient block of channel activity even at 5 uM ruthenium red (not shown). The asymmetric accessibility of ruthenium red block of reconstituted channels agrees with the data obtained from MmPiezol-overexpressing HEK293T cells (Fig. 2 and Supplementary Fig. 2), thereby establishing the fidelity of the assays and validating MmPiezol protein as an authentic ion channel. The Piezo currents exhibit ohmic behaviour; records displayed at higher resolution (Fig. 5b) clearly demonstrate the occurrence of unitary events with y values obtained from conductance histograms of 118 ± 15pS and 80 ± 6pS (n = 6) in symmetric 0.5M KC1 from the negative and positive branches of I-V plots, respectively (Fig. 5d, e).
A similar pattern of activity was obtained from MmPiezol reconstituted in asolectin liposomes26 (Fig. 5f-k). A selection of recordings shows the presence of two channels in the membrane which reside predominantly in the open state (Fig. 5f, g), as discerned in a higher time resolution display (Fig. 5k). These recordings were obtained in the presence of 50 uM ruthenium red inside the recording pipette, to ensure functional selection of a single population of MmPiezol channels facing the ruthenium-red-free compartment. MmPiezol in asolectin proteoliposomes under these conditions (symmetric 0.2 M KC1) exhibits a y = 110 ± lOpS at V= -100mV and 80 ± 5 pS at V= lOOmV (Fig. 5h-j) (n = 8). Finally, reconstitution of control samples purified from non-transfected cells as well as heat-denatured purified MmPiezol-GST into either bilayer systems under otherwise identical conditions failed to reproduce this pattern of channel activity (not shown).
We then tested the ability of the reconstituted MmPiezol to conduct sodium (Fig. 5l-q). Initially, single channel currents were recorded from asymmetric bilayers in symmetric 0.2 M KC1; y = 58 ± 5 pS (Fig. 51, o). Subsequent addition of 0.2 M NaCl in the presence of 0.2 M KC1 increased the unitary conductance of reconstituted channels to 95 ± 5 pS (Fig. 5m, p) while retaining sensitivity to ruthenium red
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■	a
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b„.      Ca2+ channel 3 1,000 t
NMDA receptor CNG channel 1,500-1 4,0001
MmPiezol
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0 10 20 30 40 Time (s)
1.0-
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Figure 4 | MmPiezol forms homo-oligomers. a, Representative image of an acquired sequence showing three selected GFP-MmPiezol spots in the cell membrane. Levels were adjusted for clarity. Scale bar, 0.8 um. b, Representative traces of fluorescence intensities of indicated single GFP-fusion constructs. Black arrows indicate photobleaching steps, c, Histograms of the average number of bleaching steps observed in ten or more movies from four or more oocytes of single fluorescent complexes of indicated constructs, d, e, Indicated samples purified and separated on native gels and visualized by Coomassie staining (d) or western blotting (e). Asterisk in d indicates a protein band
specifically present in the MmPiezol sample, f, Purified MmPiezol-GST proteins treated with or without formaldehyde (FA) with the indicated time period, separated on a denaturing gel and detected with the anti-MmPiezol antibody. Sample purified from cells without transfection served as a negative control, g, h, MmPiezol-GST-transfected HEK293T cells or untransfected cells treated with or without 0.25% PFA for 10 min. The crosslinked MmPiezol-GST proteins were purified and separated on native gel (g) or denaturing gels (h), followed by western blotting. Panels d-h are representatives of at least three independent experiments.
block (Fig. 5n, q). These results confirm that these channels conduct both sodium and potassium as would be expected from a cationic nonselective channel. This assertion was further substantiated by recording MmPiezol currents from proteoliposomes under bi-ionic conditions (0.2 M KC1/0.2 M NaCl) (Supplementary Fig. 5a-h). A summary of the I— V relation for the MmPiezol channel, extracted from 204,088 events obtained in three experiments, shows that the single channel current is ohmic between —100 and 200 mV with a slope conductance of 102 ± 2 pS (Supplementary Fig. 5i). The current reversed direction at 0.0 ± 0.3 mV, demonstrating that the channel does not select between K+ and Na+, and importantly, displays open channel block by ruthenium red (Supplementary Fig. 5j-l).
The difference in y between overexpressed MmPiezol in cells and reconstituted MmPiezol in lipid bilayers may be attributed to many variables, including the distinct lipid environments which are known to influence conductance measurements strongly2832. Moreover, the ionic conditions used in the two systems are different, as divalent cations present in HEK293T cell-attached experiments also affect the conductance values. Indeed, when divalent cations are excluded from the recording pipette, y of MmPiezol-induced currents in HEK293T cells is 58.0 pS ± 1.5 pS (150 mM NaCl solution, Supplementary Fig. 6), compared to 29.9 ± 1.5 pS in the presence of divalent ions (Fig. 3). The near equivalence of y values together with the similar pattern of channel activity demonstrates that reconstitution
of MmPiezol into two distinct bilayer systems produces channels with identical functional properties (Supplementary Table 3).
Future reconstitution and recording of DmPiezo in lipid bilayers will show whether the difference in conductance between MmPiezol and DmPiezo arises from intrinsic properties. The membrane environment and lipid composition are known to modulate the activity of the embedded channel proteins in a drastic and deterministic manner (for example, see refs 28-32). It is not entirely surprising that the conditions to emulate the cellular environment in the reconstituted system in terms of the mechanical state of the membrane or its lipid composition have thus far been inadequate to retrieve the activation features of mechanically activated ion channels. Furthermore, the complexity of protein clusters and dynamic cytoskeletal interacting partners at the cell membrane33 introduce regulatory constraints on channel activity. Further investigation may clarify whether Piezo ion channel subunits are intrinsically mechanosensitive or use unknown interacting partners to sense membrane tension.
Concluding remarks
We provide compelling evidence to support the hypothesis that Piezo proteins are indeed ion channels. First, overexpression of DmPiezo or MmPiezol in a human cell line gives rise to mechanically activated channels with distinct biophysical and pore-related properties. Second, isolated Piezo complexes do not contain detectable amounts
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100 ms
Figure 5 MmPiezol forms ruthenium-red-sensitive ion channels.
a-e, Reconstitution of purified MmPiezol into asymmetric lipid bilayers. a, Representative single channel currents at —100 mV. The section of the recordings indicated by the red asterisk is shown in b at a 10-fold higher time resolution, c, After 35 min of recording the channel activity shown in a, injection of 50 uM ruthenium red onto the neutral facing compartment blocks MmPiezo 1 currents, d, All-event current amplitude histogram of a 6-min recording; y = 124 ± 7 pS. The total number of opening events (N) analysed was 18,424. e, Single channel I- V relationship, n = 6 experiments, f-k, Reconstitution of purified MmPiezol into asolectin proteoliposomes. Representative channel currents recorded at —100 mV (f) and +100 mV (g) in the presence of 50 uM ruthenium red inside the recording pipette. Two open channels are present in the membrane. The segment of the 15 min recording shown in g indicated by the red asterisk is displayed in k at a 25-fold higher time resolution, h, i, All-event current amplitude histograms from 30 s (h) and 15 min (i) recordings: y = 110 ± 10 pS (h) and 80 ± 5 pS (i); N was 9,938 events, j, Single channel I-V relationship, n = 8 experiments. 1-q, Representative single channel currents at —100 mV of purified MmPiezol reconstituted into asymmetric lipid bilayers in symmetric 0.2 M KC1 (1), after addition of 0.2 M NaCl (m) and after addition of 50 uM ruthenium red (n). Segments indicated by red asterisks in 1-n are displayed in panels o-q, respectively. C and O denote the closed and open states.
of other channel-like proteins. Finally, purified MmPiezol protein reconstituted into proteoliposomes and planar lipid bilayers in the absence of any other cellular components gives rise to ruthenium-red-sensitive cationic ion channel activity. The MmPiezol complex is estimated to weigh ~1.2-million Da with 120-160 transmembrane segments, being, to our knowledge, the largest plasma membrane ion channel complex identified so far.
METHODS SUMMARY
Electrophysiology. Mechanical stimulation was achieved as previously described8. Subunit counting. The preparations were imaged on an inverted Nikon Ti-E fluorescence TIRF microscope (Nikon Corporation) and imaged with a high numerical aperture objective (Nikon X100 PlanApo, NA1.49). eGFP-fusion proteins were excited with a 488-nm Coherent laser (Coherent, Inc.) and images were collected with an Andor iXon DU-897 EMCCD camera.
MmPiezol-GST purification. Cells were collected and lysed 24 h after transfec-tion, followed by an affinity purification. Initially, purification was conducted
from whole-cell lysates. Thereafter, purification was performed using the membrane fraction as starting material, resulting in significantly enhanced frequency of retrieval of channel activity after reconstitution. Untransfected cells were subjected to the same purification procedure to serve as a negative control. Purified samples were kept at 4 °C until further analysis.
Native gel electrophoresis. The purified MmPiezol-GST proteins or negative control samples were subjected to 3-12% NativePAGE Novex Bis-Tris gel for native (non-denaturing) electrophoresis according to the user manual (Invitrogen). After electrophoresis, the native gel was then either visualized by a fast Coomassie G-250 staining or transferred to a PVDF membrane for western blotting. Reconstitution in lipid bilayers and proteoliposomes. Purified MmPiezol was reconstituted into proteoliposomes by detergent dilution. Excised patches from giant asolectin proteoliposomes were used for channel recordings. Asymmetric lipid bilayers were formed using the droplet interface strategy; one monolayer was composed of l,2-diphytanoyl-SM-glycero-3 phosphocholine (DPhPC), and the other of 90% DPhPC and 10% of the negatively charged lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA) (mole/mole) (Avanti Polar Lipids).
Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.
Received 22 July; accepted 21 December 2011. Published online 19 February 2012.
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26. Santos, J. S., Grigoriev, S. M. & Montal, M. Molecular tern plate for a voltage sensor in a novel K+ channel. III. Functional reconstitution of a sensorless pore module from a prokaryotic Kv channel. J. Gen. Physiol. 132, 651-666 (2008).
27. Leventis, P. A. &Grinstein, S. The distribution and function of phosphatidylserine in cellular membranes. Annu Rev Biophys 39,407^127 (2010).
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28. Ermakov, Y. A., Kamaraju, K., Sengupta, K. & Sukharev, S. Gadolinium ions block mechanosensitivechannels by altering the packingand lateral pressureofanionic lipids. Biophys. J. 98,1018-1027 (2010).
29. Gambale, F. & Montal, M. Characterization of the channel properties of tetanus toxin in planar lipid bilayers. Biophys. J. 53, 771-783 (1988).
30. Oliver, D. etal. Functional conversion between A-type and delayed rectifier K+ channels by membrane lipids. Science 304, 265-270 (2004).
31. Schmidt D. & MacKinnon, R. Voltage-dependent K+ channel gating and voltage sensor toxin sensitivity depend on the mechanical state of the lipid membrane. Proc. Natl Acad. Sci. USA 105,19276-19281 (2008).
32. Tao, X. & MacKinnon, R. Functional analysis of Kvl.2 and paddle chimera Kv channels in planar lipid bilayers. J. Mol. Biol. 382, 24-33 (2008).
33. Hartman, N. C. & Groves, J. T. Signaling clusters in the cell membrane. Curr. Opin. Cell Biol. 23,370-376 (2011).
Supplementary Information is linked to the online version of the paper at www.nature.com/nature.
Acknowledgements We thank M. H. Ulbrich for providing Ca2+ channel-, NMDA receptor-and CNG channel-GFP fusion constructs used as controls for photobleaching
experiments. This research was supported by grants from the National Institutes of Dental and Craniofacial Research, Neurological Disorders, General Medical Sciences, and by The Genomics Institute of the Novartis Research Foundation. B.X. and J.G. are postdoctoral fellowship recipients from the American Heart Association and the NIH respectively.
Author Contributions B.C. performed and analysed electrophysiological experiments. B.X. performed and analysed biochemical experiments. J.S.S. and R.S. performed the reconstitution experiments and together with M.M. analysed the single channel data. J.G. and K.S.S. performed and analysed photo-bleachingexperiments. S.E.K. cloned the Dmp/ezo gene. M.S. initiated biochemical experiments. J.M. generated GFP-MmP/ezoJ and the mRNA used for oocyte injection. A.E.D. provided technical help for oocyte experiments. A.P., B.C., B.X., J.G., J.S.S., R.S., and M.M. wrote the manuscript.
Author Information The DmPiezo sequence has been deposited in GenBank under accession number JQ425255. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www.nature.com/nature. Correspondence and requests for materials should be addressed to A.P. (apatapou@gnf.org) and M.M. (mmontal@ucsd.edu).
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RESEARCH
METHODS
Cloning of Drosophila piezo full-length cDNA. The Drosophila piezo gene (GenBank accession number JQ425255) was cloned from adult Drosophila poly(A)+ RNAs (Clonetech) by RT-PCR. Primers for RT-PCR were designed based on the annotated sequence of CG8486. Two fragments of 2 kb and 6.5 kb were amplified and cloned sequentially into pIRES2-EGFP expression vector. Each cloning step was sequence verified. Full-length Drosophila piezo gene is 8,355 bp in length. The protein sequence of DmPiezo is shown in Supplementary Fig. 1.
Cell culture and transient transfection. Human embryonic kidney 293T (HEK293T), NIH-3T3, Fll and HeLa cells were grown in Dulbecco's Modified Eagle Medium containing 4.5mgml 1 glucose, 10% fetal bovine serum, 50Uml 1 penicillin and 50|igml 1 streptomycin. Cells were plated onto poly-lysine-coated 12-mm round glass coverslips placed in 24-well plates and transfected using lipofectamine 2000 (Invitrogen) according to the manufacturer s instruction. 500-1,000 ng mP1 of plasmid DNA was transfected and cells were recorded 12-48 h later.
Electrophysiology. Patch-clamp experiments were performed in standard whole-cell or cell-attached recordings using an Axopatch 200B amplifier (Axon Instruments). Patch pipettes had resistance of 2-3 MD when filled with an internal solution consisting of (in mM) 133CsCl, 10HEPES, 5EGTA, lCaCl2, 1 MgCl2, 4 MgATP and 0.4 Na2GTP (pH adjusted to 7.3 with CsOH). The extracellular solution consisted of (in mM) 130NaCl, 3KC1, 1 MgCl2, 10HEPES, 2.5 CaCl2, 10 glucose (pH adjusted to 7.3 with NaOH). For cell-attached recordings, pipettes were filled with a solution consisting of (in mM) 130NaCl, 5KC1, 10HEPES, lCaCl2, 1 MgCl2, 10TEA-C1 (pH7.3 with NaOH), except for Supplementary Fig. 6 where the internal solution was (in mM) 150NaCl, 10HEPES (pH adjusted to 7.3 with NaOH). External solution used to zero the membrane potential consisted of (in mM) 140 KC1, 10HEPES, lMgCl2, 10 glucose (pH 7.3 with KOH). All experiments were done at room temperature. Currents were sampled at 50 or 20 kHz and filtered at 5 or 2 kHz. Voltages were not corrected for a liquid junction potential. Leak currents before mechanical stimulations were subtracted off-line from the current traces. 10 mM ruthenium red stock solution was prepared in water.
Mechanical stimulation. For whole-cell recordings mechanical stimulation was achieved using a fire-polished glass pipette (tip diameter 3-4 um) positioned at an angle of 80° to the cell being recorded. Downward movement of the probe towards the cell was driven by a Clampex controlled piezoelectric crystal micro-stage (E625 LVPZT Controller/Amplifier; Physik Instrumente). The probe was typically positioned ~2 urn from the cell body. The probe had a velocity of 1 um ms_1 during the ramp segment of the command for forward motion and the stimulus was applied for 150 ms. To assess the mechanical sensitivity of a cell, a series of mechanical steps in 1 um increments was applied every 10-20 s, which allowed full recovery of mechanosensitive currents. Inward mechanically activated currents were recorded at a holding potential of — 80mV. For I-V relationship recordings, voltage steps were applied 0.7 s before the mechanical stimulation from a holding potential of —60 mV.
For cell-attached recordings, membrane patches were stimulated with brief negative pressure pulses through the recording electrode using a Clampex controlled pressure clamp HSPC-1 device (ALA-scientific). Unless otherwise stated, stretch-activated channels were recorded at a holding potential of —80 mV with pressure steps from 0 to —60mmHg (— lOmmHg increments). Current-pressure relationships were fitted with a Boltzmann equation of the form: I(P) = [1 + exp(—(P - P50)/s)] \ where I is the peak of stretch-activated current at a given pressure, P is the applied patch pressure (in mm Hg), P50 is the pressure value that evoked a current value which is 50% of Imax, and s reflects the current sensitivity to pressure.
Single-channel amplitude characterization was performed on patches that showed strong stretch-activated current activity at —80 mV using increasing steps of negative pressure up to — 60mmHg. Similar activity was never present in control-transfected cells. Negative pressure steps were then reduced to low to moderate level (—5 to —20 mm Hg) allowing detection of single channel openings. Subunit counting. For oocyte injection, all construct plasmids were linearized at C terminus with Nhel, Hindlll or NotI and DNA transcribed with T7 mMessage mMachine Kit (Ambion) and poly(A)-tailing Kit (Ambion) and cleaned with LiCl precipitation. 50 nl of 0.2 ug ul 1 mRNA was injected into Xenopus oocytes (Nasco).
For acquisition, 12-24 h after injection, oocytes were osmotically shocked in stripping buffer (in mM: 220 N-methyl glucamine aspartate, 10 HEPES, 1 MgCl2) and mechanically de-vitellinated. MatTek dishes (MatTek Corporation) were prepared by sonication in 1M KOH to remove background fluorescence and further sonicated in MilliQ dH20. Oocytes were placed onto MatTek dishes into SOS buffer (in mM: 100 NaCl, 2 KC1, 1.8 CaCl2-H20, 1 MgCl2-6H20, 5 HEPES,
2.5 Na pyruvate and 50ugml 1 gentamicin, pH7.0). The preparations were imaged on an inverted Nikon Ti-E fluorescence TIRF microscope (Nikon Corporation) and imaged with a high numerical aperture objective (Nikon 100X PlanApo, NA1.49) with an additional X 1.5 Optovar magnification. eGFP fusion proteins were excited with a 488-nm Coherent laser (Coherent, Inc.) and images were collected with an Andor iXon DU-897 EMCCD camera. Sixty-second movies were collected at 100-ms exposures, for a frame rate of 10 Hz.
Using Nikon Elements software, movies were duplicated and processed with a rolling average of 2. A second duplicate was filtered with a low-pass kernel of 7, to remove background. The low-pass images were subtracted from the averaged images, to produce the movies used for analysis. Non-overlapping 4X4 pixel regions of interest were drawn around randomly selected spots that were clearly separated from neighbouring bright pixels. The spots were required to fit entirely within the 4X4 pixel regions. Pixel size was 0.11 um. The average intensity of each region was plotted over the length of the movies. Traces were discarded if the intensity increased after an initial decrease, if the fluorescent spot moved out of the region, or if the fluorescent signal showed a continuous decay instead of stepwise bleaching. Finally, the number of bleaching steps was counted for each spot. MmPiezol-GST purification. The MmPiezol-GST construct was subcloned by inserting a GST encoding sequence from Schistosoma japonicum into the MmPiezol construct8 at the 3' end of MmPiezol cDNA sequence using the AscI and SacII restriction enzyme sites. The resulting MmPiezol-GST fusion protein has 2,773 amino acids.
After incubation with cell lysates overnight at 4 °C, the glutathione beads were washed four times in a buffer containing 25 mM NaPIPES, 140 mM NaCl, 0.6% CHAPS, 0.14% phosphatidylcholine (PC), 2.5 mM dithiothreitol (DTT), and a cocktail of protease inhibitors and eluted with 100 mM glutathione in a buffer containing 25 mM NaPIPES, 50 mM Tris, 0.6% CHAPS, 0.14% PC, 2.5 mM DTT and a cocktail of protease inhibitors. The eluant was dialysed against a buffer containing 25 mM NaPIPES, 0.6% CHAPS, 0.14% PC, 2.5 mM DTT and a cocktail of protease inhibitors. The purified samples were kept at 4 °C. Samples purified according to this protocol were used for all the biochemical work and the initial reconstitution experiments. However, because retrieval of channel activity from the reconstituted MmPiezol-GST fluctuated from preparation to preparation, we adopted an alternative purification protocol involving the membrane fraction as the starting material. Specifically, 24 h after transfection, cells were collected and homogenized in a buffer containing 25 mM NaPIPES, 50 mM NaCl, 2.5 mM DTT, and a cocktail of protease inhibitors. The cell suspension was forced to go through a 25.5 G needle for 20 times and centrifuged at l,000g for 15min at 4°C The supernatant was collected and centrifuged at 167,000g for 30min at 4°C. The resulting membrane fraction was washed three times (using a buffer containing 25 mM NaPIPES, 150 mM NaCl, 2.5 mM DTT, and a cocktail of protease inhibitors) and used as the starting material for MmPiezol-GST purification using the same procedure described above. Purification from the membrane fraction greatly reduced the content of endogenous GST proteins and significantly enhanced the frequency of retrieval of MmPiezol channel activity after reconstitution (Fig. 5, Supplementary Fig. 5 and Supplementary Table 3).
NativePAGE Novex Bis-Tris gel. The purified MmPiezol-GST proteins and control samples were subjected to 3-12% NativePAGE Novex Bis-Tris gel for native (non-denaturing) electrophoresis according to the User Manual (Invitrogen). In brief, samples were mixed with NativePAGE Sample Buffer and NativePAGE 5% G-250 Sample Additive and then subjected to electrophoresis at 150 V for 2h. The use of G-250 charge-shift in NativePAGE gels results in protein resolution based upon protein size and therefore allows accurate size estimation of native protein complexes34. However, the native protein conformation may give an expected size estimation error of ~ 15%. After electrophoresis, the native gel was then either visualized by a fast Coomassie G-250 staining or transferred to a PVDF membrane for western blotting with an antibody specifically against Piezo 1 proteins.
Formaldehyde and paraformaldehyde crosslinking. The purified MmPiezol-GST proteins were treated with or without 0.1% formaldehyde at room temperature for different periods of time and then mixed with NuPAGE LDS Sample Buffer and NuPAGE Reducing Agent (Invitrogen), followed by heating at 70 °C for lOmin to denature the protein. The treated samples were subjected to 3-8% NuPAGE Tris-Acetate gel electrophoresis under denaturing conditions. For live cell crosslinking, 0.25% concentration of PFA was added to the cell culture medium and kept at room temperature for lOmin, followed by adding 125 mM glycine to stop the PFA crosslinking reaction. Treated cells were collected and subjected to sequential steps of protein purification, 3-8% NuPAGE Tris-Acetate gel electrophoresis under denaturing conditions or 3-12% NativePAGE Novex Bis-Tris gel for native (non-denaturing) electrophoresis, and western blotting with the anti-Piezol antibody.
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Western blotting. After electrophoresis, either the native or denaturing PAGE gels were transferred to PVDF membranes. Transferring protein from native gel to PVDF membranes was conducted according to instructions for NativePAGE Novex Bis-Tris gel system. Transferred PVDF membranes were blocked with 5% milk in TBS buffer with 0.1% Tween-20 (TBST buffer) at room temperature for 1 h, and then incubated with the anti-Piezol antibody (1:200) at 4 °C overnight. The membranes were washed with TBST buffer and incubated with peroxidase-conjugated anti-rabbit IgG secondary antibody (1:10,000) at room temperature for 1 h. Proteins were detected with the ECL plus detection kit (GE Healthcare). Mass spectrometry. Purified samples were separated on the 3-12% NativePAGE Novex Bis-Tris gel and visualized by fast Coomassie G-250 staining. The gel band containing the MmPiezol-GST complex or the corresponding blank band from the control sample near the 1,236 kDa molecular marker was excised and subjected to the Scripps Center for Mass Spectrometry for analysis. In brief, the gel bands were destained, reduced with 10 mM DTT, alkylated with 55 mM iodoacetamide, and digested with Trypsin overnight before analysis using the nano-LC-MS/MS. The nano-LC-MS/MS data obtained on a LTQ ion trap mass spectrometer was searched using the MmPiezol-GST protein sequence and NCBInr Homo sapiens database. In separate sets of experiments, the purified MmPiezol-GST and control solution samples before gel electrophoresis were subjected to mass spectrometry (Supplementary Table 2). Reconstitution into proteoliposomes or DIBs, single channel recordings and analysis. Purified MmPiezol-GST protein was reconstituted into asolectin (soybean polar lipid extract, Avanti) liposomes (lOmgml l) by incubating the mixture (lipid/protein mass ratios between 2,000:1 and 1,000:1; this corresponds to a molar lipid/protein ratio of ~800,000-400,000:1) on ice for 5 min followed by X20 dilution in 200 mM KC1,5 mM MOPS pH 7.0 and incubated with rotation at room temperature for 20 min. Biobeads were added to mixture and incubated with rotation for 1 h. Thereafter, biobeads were removed by filtration and a new batch of beads was added. After 30 min incubation, the biobeads were filtered and the sample was centrifuged at 60,000 r.p.m. for 60 min at 8 °C. The proteoliposome pellet was re-suspended in 40 ul of the same buffer and used to place two 25 ul drops on a cover slide. The samples were dried under vacuum for > 16 h at 4 °C. Samples were hydrated with 25 ul of the same buffer and allowed to sit for 2 h before starting recordings. Thereafter, 2-3 ul of proteoliposomes were withdrawn from the edge of the spots on the cover slide and transferred to the recording chamber. After 5 min, the chamber was slowly filled with recording solution. Multi-G£2 seals were made to proteoliposomes immobilized at the bottom of the
recording chamber. At that time, the proteoliposome patch was excised and brought through the air-water interface. Excised patches were used35. Pipette and bath solution contained (in mM) 200 KC1, 5 MOPS titrated to pH 7.0 with KOH. Capillaries of borosilicate glass from Sigma were pulled to yield resistances of 1-2 MQ when immersed in recording solution.
Droplet interface lipid bilayers (DIBs) were formed between two lipid monolayer-encased aqueous nanolitre droplets submerged in hexadecane23. Liposomes were composed of l,2-diphytanoyl-sn-glycero-3 phosphocholine (DPhPC) or 90% (mole/mole) DPhPC and 10% of the negatively charged lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA) (Avanti Polar Lipids). MmPiezol was diluted directly into the liposome suspension to yield a final concentration of 2-5ngml l. The electrode carrying the droplet with MmPiezol and desired buffer-lipid mix (in mM, 500 KC1, 10 HEPES, pH7.4, 0.5 lipid solution of DPhPC) was connected to the grounded end of the amplifier head-stage (Axopatch 200B). The second electrode, in a droplet containing the same buffer and 10% DOPA:90% DPhPC, was connected to the working end of the head-stage. Where indicated, ruthenium red or 0.2 M NaCl was injected using a nano-injector (WPI, Inc.).
For proteoliposome patches, records were acquired at a sampling frequency of 40 kHz and filtered online to 5 kHz with a 3-pole Bessel filter before digitization; for DIBs, data acquisition was at 10 kHz and filtered at 2 kHz. For analysis and presentation, records were filtered to 1 kHz with a low-pass Gaussian filter. Transitions were detected by the half-threshold method implemented in Clampfit (proteoliposomes) and by the segmental fc-means method (SKM) implemented in QuB (DIBs). Transitions £0.5 ms were excluded from the pool for analysis to correct for detection of false and missed events. Data were analysed using Clampfit v.9.2 software (Axon Instruments), QuB, Excel 2007 (Microsoft), and IGOR Pro (Wavemetrics). y was calculated from Gaussian fits to currents histograms. All statistical values represent mean ± s.e.m., unless otherwise indicated, n and N denote number of experiments and number of events, respectively. All experiments were done at room temperature.
34. Schagger, H., Cramer, W. A. & von Jagow, G. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal. Biochem. 217, 220-230 (1994).
35. Gam bale, F. & Montal, M. Voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671. Brain Res. Mol. Brain Res. 7,123-129 (1990).
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