- Email: [email protected]
Heterologous Expression of the Piezo1-ASIC1 Chimera Induces Mechanosensitive Currents with Properties Distinct from Piezo1
Matters Arising Response
Heterologous Expression of the Piezo1-ASIC1 Chimera Induces Mechanosensitive Currents with Properties Distinct from Piezo1 Highlights
Authors
d
Piezo1(1–2190)-ASIC1 mediates mechanosensitive currents in HEK293T cells
Qiancheng Zhao, Kun Wu, Shaopeng Chi, Jie Geng, Bailong Xiao
d
Piezo1(1–2190)-ASIC1 displays current properties distinct from Piezo1
Correspondence
HEK293T cells have nearly no detectable expression of endogenous Piezo1
In Brief
d
d
The chimera-induced currents are unlikely to be directly due to endogenous Piezo1
Zhao et al., 2017, Neuron 94, 274–277 April 19, 2017 ª 2017 Elsevier Inc. http://dx.doi.org/10.1016/j.neuron.2017.03.040
[email protected]
Zhao et al. have shown that fusing the N-terminal non-pore-containing region (1–2190) of Piezo1 to the mechanoinsensitive ASIC1 channel generates a chimeric protein, Piezo1(1–2190)-ASIC1, that consistently induces mechanosensitive currents in HEK293T cells with properties distinct from endogenous Piezo1-mediated currents.
Neuron
Matters Arising Response Heterologous Expression of the Piezo1-ASIC1 Chimera Induces Mechanosensitive Currents with Properties Distinct from Piezo1 Qiancheng Zhao,1 Kun Wu,1 Shaopeng Chi,1 Jie Geng,1 and Bailong Xiao1,2,* 1School of Pharmaceutical Sciences, Tsinghua-Peking Joint Center for Life Sciences, IDG/McGovern Institute for Brain Research, Tsinghua University, Beijing 100084, China 2Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2017.03.040
SUMMARY
Piezo1 represents a prototype of the mammalian mechanosensitive cation channel, but its molecular mechanism remains elusive. In a recent study, we showed that C-terminal region, which contains the last two TMs, of 2189–2547 of Piezo1 forms the bona fide pore module, and systematically identified the pore-lining helix and key poreproperty-determining residues (Zhao et al., 2016). Furthermore, we have engineered the Piezo1(1– 2190)-ASIC1 chimera (fusing the N-terminal region of 1–2190 to the mechano-insensitive ASIC1) that mediated mechanical- and acid-evoked currents in HEK293T cells, indicating the sufficiency of the N-terminal region in mechanotransduction. Now in a Matters Arising, the authors specifically questioned the implication of the chimera data among the many findings shown in our paper. They replicated the chimera-mediated mechanosensitive currents in HEK293T cells that have nearly no detectable expression of endogenous Piezo1, but paradoxically found the chimera to be less effective in Piezo1 knockout HEK293T cells, indicating the involvement of endogenous Piezo1. In this Matters Arising Response, we discuss the chimera results and consider potential interpretations in light of the Matters Arising from Dubin et al. (2017), published concurrently in this issue of Neuron. Please see also the response from Hong et al. (2017), published in this issue. INTRODUCTION The Piezo family of proteins, including Piezo1 and Piezo2, has been identified as a bona fide class of mechanosensitive cation channels that play critical roles in various mechanotransduction processes (Coste et al., 2010, 2012; Ranade et al., 2015). Thus, it is imperative to understand how Piezo proteins function as a distinct class of mechanosensitive cation channels. 274 Neuron 94, 274–277, April 19, 2017 ª 2017 Elsevier Inc.
In a recent study reported in Neuron (Zhao et al., 2016), we systematically investigated the molecular bases of the ionconducting pathway and mechanotransduction mechanisms of the Piezo1 channel, which enabled us for the first time to functionally identify the bona fide ion-permeating pore and mechanotransduction component of this novel and distinct class of ion channel. Our major findings include the following: (1) The very C-terminal region, which contains the last two transmembrane segments (TMs), of residues 2189–2547 encodes the pore-forming module of Piezo1 that resembles the pore architecture of other trimeric channels such as the acid-sensing ion channel 1 (ASIC1) and the ATP-gated P2X4 channel, and determines the essential pore properties. (2) A cluster of negatively charged residues (DEEED (2393–2397)) located at the fenestration sites of the C-terminal extracellular domain (CED)-constituted extracellular ‘‘Cap’’ structure is critical for selecting cation over anion and efficient ion conduction. (3) The last putative TM forms the pore-lining helix. (4) The acidic residues, E2495 and E2496, located in the intracellular C-terminal domain (CTD) constitute the key pore determinants that determine single-channel conductance, ion selectivity, and pore blockade. (5) Fusing the N-terminal region of 1–2190 of Piezo1 to the mechano-insensitive ASIC1 resulted in a chimeric channel (Piezo1(1–2190)-ASIC1) that responded to both acid and mechanical stimulation, and was sensitive to ASIC1 channel blocker amiloride. These data suggest that the peripheral propeller structure formed by the N-terminal non-pore-containing region in principle might be sufficient to confer mechanosensitivity to Piezo1 channels. Collectively, these functional characterizations are consistent with structural organization of mPiezo1 into a unique three-bladed, propeller-shaped architecture comprising the central ion-conducting pore module and the three peripheral propeller structures that might serve as mechanotransduction modules (Ge et al., 2015). Now in the Matters Arising, the authors specifically question the fifth finding listed above and shown in our paper (Zhao et al., 2016). Importantly, they replicated our results that a portion of HEK293T cells transfected with the Piezo1(1–2190)ASIC1 chimera showed significantly enhanced mechanically activated (MA) currents (16 out of 34 cells showing MA currents). However, when they carried out new experiments using a HEK293T cell line in which endogenous Piezo1 was truncated
Table 1. Summary of the Mechanosensitivity of the Engineered Chimeric Constructs Chimeras
GGGGG Linker
MA Currents
Piezo1(1–2170)-ASIC1
yes
no
Piezo1(1–2181)-ASIC1
yes
no
*Piezo1(1–2190)-ASIC1
yes
yes
Piezo1(1–2190)-ASIC1
no
no
Piezo1(1–2190)-P2X4
yes
no
Chimeric constructs were generated to test the sufficiency of the N-terminal non-pore-containing regions of Piezo1 for conferring mechanosensitivity to mechano-insensitive trimeric channels, including ASIC1 and P2X4. The construct marked with an asterisk (*) is the only chimera that exhibited mechanically activated (MA) currents when heterologously expressed. The success for generating the MA chimera is dependent on the precise N-terminal sequence composition of Piezo1 (only 1–2190), the specific trimeric channel pore (only ASIC1, but not P2X4), and the GGGGGG linker in between, suggesting that the MA current observed in Piezo1(1–2190)-ASIC1 (with the GGGGG linker) overexpressed HEK293T cells is a specific response to the chimera.
near K1984 of human Piezo1 (corresponding to K2000 of mouse Piezo1) using CRISPR/Cas9 (HEK-P1 knockout, Clone 1C10), they detected 1 out of 29 cells generating MA currents. No MA current was detected from vector-transfected HEK-P1 knockout cells. Based on this study, they have suggested that the MA currents observed in the chimera-transfected wild-type HEK293T cells are due to endogenous Piezo1. We appreciate the use of the HEK-P1 knockout cell line as a heterologous expression system for further characterizing the mechanosensitivity of the chimeric channel, and agree that the new study might lead to an alternative interpretation of the Piezo1(1–2190)-ASIC1 chimera results shown in our paper. However, given that HEK293T cells express nearly no detectable level of endogenous Piezo1 and that the chimera-mediated currents are clearly different from Piezo1dependent currents, we consider that this evidence appears not to be consistent with the suggestion that the chimeramediated currents in HEK293T cells are due to endogenous Piezo1. Therefore, we have also raised other potential scenarios that might account for the lesser effectiveness of the chimera in mediating mechanosensitive currents in the HEK-P1 knockout cells. RESULTS AND DISCUSSION Piezo1(1–2190)-ASIC1, but Not Other Chimeras, Mediates MA Currents in HEK293T Cells We have engineered a series of chimeric mutants by fusing the N-terminal non-pore-containing regions (1–2170, 1–2181, and 1–2190) of Piezo1 with ASIC1 (with a GGGGG linker in between) (Table 1). Intriguingly, we found that only the chimeric construct Piezo1(1–2190)-ASIC1 gave rise to a portion of the transfected HEK293T cells with MA currents, which is statistically significantly different from ASIC1-transfected cells (Figures 7A and 7B in Zhao et al., 2016). By contrast, other chimeric constructs, including Piezo1(1–2170)-ASIC1, Piezo1(1–2181)ASIC1, and Piezo1(1–2190)-ASIC1 (without the GGGGG linker),
did not show MA currents (Table 1). Furthermore, the chimera (Piezo1(1–2190)-P2X4 [with a GGGGG linker in between]) also failed to give rise to MA currents (Table 1). These data suggest that the MA currents specifically observed in the Piezo1(1– 2190)-ASIC1-transfected HEK293T cells are unlikely due to transfection-induced alteration of the cell state, and that the linker region between the N-terminal fragment of Piezo1 and ASIC1 appears to be critical. Piezo1(1–2190)-ASIC1 Shows Channel Properties Distinct from Piezo1 Our characterization of the Piezo1(1–2190)-ASIC1-mediated MA currents in HEK293T cells suggest that the chimera has current properties clearly distinct from wild-type Piezo1. These distinct properties include the following: (1) The minimum displacement of the probing pipette to induce MA currents (probing threshold) in Piezo1(1–2190)-ASIC1-transfected cells is increased compared to Piezo1-transfected cells (8.81 ± 0.90 mm versus 5.25 ± 0.52 mm, respectively) (Figure 7B in Zhao et al., 2016), indicating compromised mechanotransduction efficiency in the chimeric channel. (2) The chimera-mediated MA currents show apparent inactivation, but with a slower inactivation tau than that of wild-type Piezo1 (21.0 ± 2.5 ms versus 16.0 ± 1.2 ms, respectively). (3) A portion of the Piezo1(1–2190)ASIC1-transfected cells also responded to acid (Figures 7C and 7D in Zhao et al., 2016). Given that previous studies have shown that Piezo1 channels are inhibited by acid (Bae et al., 2015), these data suggest that the mechanical force- or acid-induced current in Piezo1(1–2190)-ASIC1-transfected cells is mediated by the ASIC channel pore of the chimera protein. (4) The MA currents of the chimera were partially blocked by amiloride at 100 mM concentration that effectively blocked ASIC1, but not Piezo1 (Figures 7E–7H in Zhao et al., 2016), further supporting the idea that the ion-conducting pore is distinct from the Piezo1 channel pore. Notably, the blocking effect of amiloride on the chimeric channel was less efficient than on ASIC1 (49.7% ± 9.0% versus 14.1% ± 5.2%, respectively) (Figures 7E–7H in Zhao et al., 2016), suggesting that attachment of the bulky N-terminal region of mPiezo1 might alter the pore properties of ASIC1. (5) The reversal potential of the chimera was significantly different from that of the sodium-selective ASIC1 and non-selective mPiezo1, indicating that the MA current of the chimeric channel was non-selective for Na+ over K+ despite the presence of the ASIC1 channel pore (Figures S5D–S5F in Zhao et al., 2016). Given that ASIC1 channels are capable of adopting both Na+-selective and non-selective conformations of their ion-conducting pore (i.e., the PcTX1 [a spider toxin]-bound ASIC1 at neutral pH forms a non-selective cation channel, while at low pH is Na+ selective) (Baconguis and Gouaux, 2012), it is not totally unexpected that the bulky N-terminal region of mPiezo1 might cause alteration of the pore properties of the fused ASIC1. We hypothesize that the ion selectivity of the Piezo1 channel pore could be flexible, which merits further investigation. Based on the characterization of the Piezo1(1–2190)-ASIC1 chimera and the structural organization of Piezo1, we suggest that the peripheral propeller structure formed by the N-terminal region of 1–2190 might in principle be sufficient to confer mechanosensitivity to Piezo1 channels. Neuron 94, 274–277, April 19, 2017 275
Evidence Inconsistent with the Suggestion that the Piezo1(1–2190)-ASIC1-Mediated Current Is Due to Endogenous Piezo1 in HEK293T Cells The expression of endogenous Piezo1 in HEK293 cells is too low to even be reliably detected by RT-PCR, which has been clearly pointed out by Dubin et al. in the Matters Arising as well. Paradoxically, they have shown that 11 out of 25 vectortransfected cells were found to have MA currents. It is not clear why the wild-type HEK293T cells in the Matters Arising have such a high percentage of MA currents. In our experience and under the experimental conditions used in the previous study (Zhao et al., 2016), the chance to detect endogenous MA currents in HEK293T cells is rare. For instance, we only detected 2 out of 20 vector-transfected HEK293 cells that had MA currents with an amplitude of about 20 pA. Previous studies by Coste et al. showed negligible MA currents from vector-transfected HEK293 cells as well (Coste et al., 2010). By contrast, the average maximal current of Piezo1(1–2190)ASIC1 is 132.5 ± 24.3 pA (Zhao et al., 2016). One characteristic feature of the endogenous MA currents of wild-type HEK293 cells shown in the Matters Arising is the apparent lack of inactivation (Figure 1 of Dubin et al., 2017). However, the chimera-mediated MA currents display clear inactivation, as shown in Figure 7 of our study (Zhao et al., 2016) as well as in Figures 2 and 3 of Dubin et al. (2017). Dubin et al. have suggested that overexpression of Piezo1(1– 2190)-ASIC1 might potentiate the expression of endogenous Piezo1. However, as described above, the chimera-mediated current is clearly distinct from the wild-type Piezo1-mediated current. Furthermore, the authors in the Matters Arising observed that the apparent threshold for responsive vectortransfected HEK293T cells (12.4 ± 1.1 mm) was about twice that observed for cells expressing the chimera (6 mm). It is striking that the threshold of the chimera is comparable to that of the exogenously expressed Piezo1 in HEK293T cells (5.8 ± 0.8 mm), as shown in the Matters Arising. In our study (Zhao et al., 2016), the thresholds for Piezo1(1–2190)ASIC1 and Piezo1 in HEK293T cells were 8.8 ± 0.90 mm and 5.25 ± 0.52 mm, respectively. Thus, both studies consistently show that the threshold of the chimera-mediated MA currents is clearly distinct from the endogenous MA currents observed in HEK293T cells by Dubin et al. Another possibility suggested by Dubin et al. is that overexpression of the Piezo1(1–2190)-ASIC1 chimera might lead to unknown stresses to cells that indirectly modulate Piezo1 function. However, this appears to be inconsistent with our observation that transfection of other chimeras differing slightly in amino acid compositions, including Piezo1(1–2170)ASIC1, Piezo1(1–2181)-ASIC1, Piezo1(1–2190)-ASIC1 (without the GGGGG linker), and Piezo1(1–2190)-P2X4 (with a GGGGG linker in between), failed to induce MA currents. Alternatively, the overexpressed Piezo1(1–2190)-ASIC1 proteins might form heteromeric channels with endogenous Piezo1. This might explain the altered current properties recorded from the chimera-transfected HEK293T cells. However, in this scenario, as the authors in the Matters Arising pointed out, the transfected construct Piezo1-ASIC1 is expected to be logs in excess of any endogenous Piezo1. The heteromeric channel, if it exists, 276 Neuron 94, 274–277, April 19, 2017
is more likely to be composed of two Piezo1(1–2190)-ASIC1 subunits and one endogenous Piezo1 subunit (assuming that the heteromeric channel also has a trimeric architecture). This would still support the idea that the N-terminal region of Piezo1 might confer mechanosensitivity to the heteromeric pore. Other Scenarios that Might Account for the Lesser Effectiveness of Piezo1(1–2190)-ASIC1 in Mediating MA Currents in the HEK-P1 Knockout Cells Currently, it is not clear why overexpression of the Piezo1(1– 2190)-ASIC1 protein in the HEK-P1 knockout cell line was less effective in generating MA currents than in HEK293T cells. One possibility is that the truncated Piezo1 proteins (1–1984) (without the C-terminal pore-forming domain) might cause dominantnegative suppression of the chimeric proteins if the chimeramediated currents observed in wild-type HEK293 cells are due to formation of heteromeric channels with the endogenous Piezo1 proteins. Another possibility is that the HEK-P1 knockout cell line might not be fully capable of supporting all mechanosensitive ion channels. Indeed, in addition to functioning as ion-permeating pores, mechanosensitive ion channels such as TREK1 have been shown to have a drastic effect on cytoskeleton and cell morphology (Lauritzen et al., 2005). Thus, it is possible that Piezo1 might underlie some roles (not necessary related to its ion-conducting pore function) in affecting some unclear aspects of cellular properties. In line with this idea, Piezo1 has been shown to be able to activate integrin and affect cell migration (McHugh et al., 2010, 2012). Indeed, Dubin et al. have noticed that the HEK-P1 knockout cell line requires much a longer time (up to four to five passages) for recovery after thawing, indicating potential abnormity and adaptation of the cells. It is expected that overexpression of Piezo1 itself in the knockout cell line should fully rescue any phenotypes due to its depletion and thus generate Piezo1-mediated MA currents. However, this does not mean that the knockout cell line might support mechanotransduction of all other mechanosensitive ion channels, including Piezo2, NOMPC, TREK, TMEM150C, and Piezo1(1– 2190)-ASIC1. Furthermore, the method used by the authors in the Matters Arising for generating the Piezo1 knockout cell line is prone to introducing non-specific mutations in the whole genome, which might cause undetected effects on the cellular properties of the cell line. For instance, what causes the observed slower recovery of the HEK-P1 knockout cell line? Thus, the chimera should be tested in different clones of HEK-P1 knockout cell lines as well as in those clones that had experienced the same selection processes during the generation of the knockout cell line, but with the Piezo1 gene remaining intact. In summary, the finding that transfection of HEK293T cells with the Piezo1(1–2190)-ASIC1 chimera can give rise to MA currents has been consistently observed. The new finding by Dubin et al. with the use of the HEK-P1 knockout cells complicates the interpretation of the implication of the chimera results. Nevertheless, together with the structural features of the Piezo1 channel (Ge et al., 2015), the hypothesis that the N-terminal propeller-resembling structure might serve as an intrinsic mechanotransduction module for the Piezo1 channel remains alive for further testing.
For instance, future studies should focus on identifying specific regions/residues in the N-terminal region that critically determine the mechanotransduction process of the Piezo1 channel. For a novel and complicated ion channel family like Piezos, it is reasonable to believe that extensive investigations need to be done in order to obtain a thorough mechanistic understanding, which might eventually allow explanation of any paradox arising during the course of the study. AUTHOR CONTRIBUTIONS B.X. wrote the paper and all authors discussed and read the paper. ACKNOWLEDGMENTS
Coste, B., Mathur, J., Schmidt, M., Earley, T.J., Ranade, S., Petrus, M.J., Dubin, A.E., and Patapoutian, A. (2010). Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330, 55–60. Coste, B., Xiao, B., Santos, J.S., Syeda, R., Grandl, J., Spencer, K.S., Kim, S.E., Schmidt, M., Mathur, J., Dubin, A.E., et al. (2012). Piezo proteins are pore-forming subunits of mechanically activated channels. Nature 483, 176–181. Dubin, A.E., Murthy, S., Lewis, A.H., Brosse, L., Cahalan, S.M., Grandl, J., Coste, B., and Patapoutian, A. (2017). Endogenous Piezo1 can confound mechanically activated channel identification and characterization. Neuron 94, this issue, 266–270. Ge, J., Li, W., Zhao, Q., Li, N., Chen, M., Zhi, P., Li, R., Gao, N., Xiao, B., and Yang, M. (2015). Architecture of the mammalian mechanosensitive Piezo1 channel. Nature 527, 64–69.
This work was supported by the National Natural Science Foundation of China (31422027, 31371118, and 31630090), the Ministry of Science and Technology (2015CB910102 and 2016YFA0500402), and the Ministry of Education (Young Thousand Talent Program) to B.X.
Hong, G.-S., Lee, B., and Oh, U. (2017). Evidence for mechanosensitive channel activity of tentonin 3/TMEM150C. Neuron 94, this issue, 271–273.
Received: December 6, 2016 Revised: January 22, 2017 Accepted: March 27, 2017 Published: April 19, 2017
McHugh, B.J., Buttery, R., Lad, Y., Banks, S., Haslett, C., and Sethi, T. (2010). Integrin activation by Fam38A uses a novel mechanism of R-Ras targeting to the endoplasmic reticulum. J. Cell Sci. 123, 51–61.
REFERENCES Baconguis, I., and Gouaux, E. (2012). Structural plasticity and dynamic selectivity of acid-sensing ion channel-spider toxin complexes. Nature 489, 400–405. Bae, C., Sachs, F., and Gottlieb, P.A. (2015). Protonation of the human PIEZO1 ion channel stabilizes inactivation. J. Biol. Chem. 290, 5167–5173.
Lauritzen, I., Chemin, J., Honore´, E., Jodar, M., Guy, N., Lazdunski, M., and Jane Patel, A. (2005). Cross-talk between the mechano-gated K2P channel TREK-1 and the actin cytoskeleton. EMBO Rep. 6, 642–648.
McHugh, B.J., Murdoch, A., Haslett, C., and Sethi, T. (2012). Loss of the integrin-activating transmembrane protein Fam38A (Piezo1) promotes a switch to a reduced integrin-dependent mode of cell migration. PLoS ONE 7, e40346. Ranade, S.S., Syeda, R., and Patapoutian, A. (2015). Mechanically activated ion channels. Neuron 87, 1162–1179. Zhao, Q., Wu, K., Geng, J., Chi, S., Wang, Y., Zhi, P., Zhang, M., and Xiao, B. (2016). Ion permeation and mechanotransduction mechanisms of mechanosensitive Piezo channels. Neuron 89, 1248–1263.
Neuron 94, 274–277, April 19, 2017 277
Heterologous Expression of the Piezo1-ASIC1 Chimera Induces Mechanosensitive Currents with Properties Distinct from Piezo1 Highlights
Authors
d
Piezo1(1–2190)-ASIC1 mediates mechanosensitive currents in HEK293T cells
Qiancheng Zhao, Kun Wu, Shaopeng Chi, Jie Geng, Bailong Xiao
d
Piezo1(1–2190)-ASIC1 displays current properties distinct from Piezo1
Correspondence
HEK293T cells have nearly no detectable expression of endogenous Piezo1
In Brief
d
d
The chimera-induced currents are unlikely to be directly due to endogenous Piezo1
Zhao et al., 2017, Neuron 94, 274–277 April 19, 2017 ª 2017 Elsevier Inc. http://dx.doi.org/10.1016/j.neuron.2017.03.040
[email protected]
Zhao et al. have shown that fusing the N-terminal non-pore-containing region (1–2190) of Piezo1 to the mechanoinsensitive ASIC1 channel generates a chimeric protein, Piezo1(1–2190)-ASIC1, that consistently induces mechanosensitive currents in HEK293T cells with properties distinct from endogenous Piezo1-mediated currents.
Neuron
Matters Arising Response Heterologous Expression of the Piezo1-ASIC1 Chimera Induces Mechanosensitive Currents with Properties Distinct from Piezo1 Qiancheng Zhao,1 Kun Wu,1 Shaopeng Chi,1 Jie Geng,1 and Bailong Xiao1,2,* 1School of Pharmaceutical Sciences, Tsinghua-Peking Joint Center for Life Sciences, IDG/McGovern Institute for Brain Research, Tsinghua University, Beijing 100084, China 2Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2017.03.040
SUMMARY
Piezo1 represents a prototype of the mammalian mechanosensitive cation channel, but its molecular mechanism remains elusive. In a recent study, we showed that C-terminal region, which contains the last two TMs, of 2189–2547 of Piezo1 forms the bona fide pore module, and systematically identified the pore-lining helix and key poreproperty-determining residues (Zhao et al., 2016). Furthermore, we have engineered the Piezo1(1– 2190)-ASIC1 chimera (fusing the N-terminal region of 1–2190 to the mechano-insensitive ASIC1) that mediated mechanical- and acid-evoked currents in HEK293T cells, indicating the sufficiency of the N-terminal region in mechanotransduction. Now in a Matters Arising, the authors specifically questioned the implication of the chimera data among the many findings shown in our paper. They replicated the chimera-mediated mechanosensitive currents in HEK293T cells that have nearly no detectable expression of endogenous Piezo1, but paradoxically found the chimera to be less effective in Piezo1 knockout HEK293T cells, indicating the involvement of endogenous Piezo1. In this Matters Arising Response, we discuss the chimera results and consider potential interpretations in light of the Matters Arising from Dubin et al. (2017), published concurrently in this issue of Neuron. Please see also the response from Hong et al. (2017), published in this issue. INTRODUCTION The Piezo family of proteins, including Piezo1 and Piezo2, has been identified as a bona fide class of mechanosensitive cation channels that play critical roles in various mechanotransduction processes (Coste et al., 2010, 2012; Ranade et al., 2015). Thus, it is imperative to understand how Piezo proteins function as a distinct class of mechanosensitive cation channels. 274 Neuron 94, 274–277, April 19, 2017 ª 2017 Elsevier Inc.
In a recent study reported in Neuron (Zhao et al., 2016), we systematically investigated the molecular bases of the ionconducting pathway and mechanotransduction mechanisms of the Piezo1 channel, which enabled us for the first time to functionally identify the bona fide ion-permeating pore and mechanotransduction component of this novel and distinct class of ion channel. Our major findings include the following: (1) The very C-terminal region, which contains the last two transmembrane segments (TMs), of residues 2189–2547 encodes the pore-forming module of Piezo1 that resembles the pore architecture of other trimeric channels such as the acid-sensing ion channel 1 (ASIC1) and the ATP-gated P2X4 channel, and determines the essential pore properties. (2) A cluster of negatively charged residues (DEEED (2393–2397)) located at the fenestration sites of the C-terminal extracellular domain (CED)-constituted extracellular ‘‘Cap’’ structure is critical for selecting cation over anion and efficient ion conduction. (3) The last putative TM forms the pore-lining helix. (4) The acidic residues, E2495 and E2496, located in the intracellular C-terminal domain (CTD) constitute the key pore determinants that determine single-channel conductance, ion selectivity, and pore blockade. (5) Fusing the N-terminal region of 1–2190 of Piezo1 to the mechano-insensitive ASIC1 resulted in a chimeric channel (Piezo1(1–2190)-ASIC1) that responded to both acid and mechanical stimulation, and was sensitive to ASIC1 channel blocker amiloride. These data suggest that the peripheral propeller structure formed by the N-terminal non-pore-containing region in principle might be sufficient to confer mechanosensitivity to Piezo1 channels. Collectively, these functional characterizations are consistent with structural organization of mPiezo1 into a unique three-bladed, propeller-shaped architecture comprising the central ion-conducting pore module and the three peripheral propeller structures that might serve as mechanotransduction modules (Ge et al., 2015). Now in the Matters Arising, the authors specifically question the fifth finding listed above and shown in our paper (Zhao et al., 2016). Importantly, they replicated our results that a portion of HEK293T cells transfected with the Piezo1(1–2190)ASIC1 chimera showed significantly enhanced mechanically activated (MA) currents (16 out of 34 cells showing MA currents). However, when they carried out new experiments using a HEK293T cell line in which endogenous Piezo1 was truncated
Table 1. Summary of the Mechanosensitivity of the Engineered Chimeric Constructs Chimeras
GGGGG Linker
MA Currents
Piezo1(1–2170)-ASIC1
yes
no
Piezo1(1–2181)-ASIC1
yes
no
*Piezo1(1–2190)-ASIC1
yes
yes
Piezo1(1–2190)-ASIC1
no
no
Piezo1(1–2190)-P2X4
yes
no
Chimeric constructs were generated to test the sufficiency of the N-terminal non-pore-containing regions of Piezo1 for conferring mechanosensitivity to mechano-insensitive trimeric channels, including ASIC1 and P2X4. The construct marked with an asterisk (*) is the only chimera that exhibited mechanically activated (MA) currents when heterologously expressed. The success for generating the MA chimera is dependent on the precise N-terminal sequence composition of Piezo1 (only 1–2190), the specific trimeric channel pore (only ASIC1, but not P2X4), and the GGGGGG linker in between, suggesting that the MA current observed in Piezo1(1–2190)-ASIC1 (with the GGGGG linker) overexpressed HEK293T cells is a specific response to the chimera.
near K1984 of human Piezo1 (corresponding to K2000 of mouse Piezo1) using CRISPR/Cas9 (HEK-P1 knockout, Clone 1C10), they detected 1 out of 29 cells generating MA currents. No MA current was detected from vector-transfected HEK-P1 knockout cells. Based on this study, they have suggested that the MA currents observed in the chimera-transfected wild-type HEK293T cells are due to endogenous Piezo1. We appreciate the use of the HEK-P1 knockout cell line as a heterologous expression system for further characterizing the mechanosensitivity of the chimeric channel, and agree that the new study might lead to an alternative interpretation of the Piezo1(1–2190)-ASIC1 chimera results shown in our paper. However, given that HEK293T cells express nearly no detectable level of endogenous Piezo1 and that the chimera-mediated currents are clearly different from Piezo1dependent currents, we consider that this evidence appears not to be consistent with the suggestion that the chimeramediated currents in HEK293T cells are due to endogenous Piezo1. Therefore, we have also raised other potential scenarios that might account for the lesser effectiveness of the chimera in mediating mechanosensitive currents in the HEK-P1 knockout cells. RESULTS AND DISCUSSION Piezo1(1–2190)-ASIC1, but Not Other Chimeras, Mediates MA Currents in HEK293T Cells We have engineered a series of chimeric mutants by fusing the N-terminal non-pore-containing regions (1–2170, 1–2181, and 1–2190) of Piezo1 with ASIC1 (with a GGGGG linker in between) (Table 1). Intriguingly, we found that only the chimeric construct Piezo1(1–2190)-ASIC1 gave rise to a portion of the transfected HEK293T cells with MA currents, which is statistically significantly different from ASIC1-transfected cells (Figures 7A and 7B in Zhao et al., 2016). By contrast, other chimeric constructs, including Piezo1(1–2170)-ASIC1, Piezo1(1–2181)ASIC1, and Piezo1(1–2190)-ASIC1 (without the GGGGG linker),
did not show MA currents (Table 1). Furthermore, the chimera (Piezo1(1–2190)-P2X4 [with a GGGGG linker in between]) also failed to give rise to MA currents (Table 1). These data suggest that the MA currents specifically observed in the Piezo1(1– 2190)-ASIC1-transfected HEK293T cells are unlikely due to transfection-induced alteration of the cell state, and that the linker region between the N-terminal fragment of Piezo1 and ASIC1 appears to be critical. Piezo1(1–2190)-ASIC1 Shows Channel Properties Distinct from Piezo1 Our characterization of the Piezo1(1–2190)-ASIC1-mediated MA currents in HEK293T cells suggest that the chimera has current properties clearly distinct from wild-type Piezo1. These distinct properties include the following: (1) The minimum displacement of the probing pipette to induce MA currents (probing threshold) in Piezo1(1–2190)-ASIC1-transfected cells is increased compared to Piezo1-transfected cells (8.81 ± 0.90 mm versus 5.25 ± 0.52 mm, respectively) (Figure 7B in Zhao et al., 2016), indicating compromised mechanotransduction efficiency in the chimeric channel. (2) The chimera-mediated MA currents show apparent inactivation, but with a slower inactivation tau than that of wild-type Piezo1 (21.0 ± 2.5 ms versus 16.0 ± 1.2 ms, respectively). (3) A portion of the Piezo1(1–2190)ASIC1-transfected cells also responded to acid (Figures 7C and 7D in Zhao et al., 2016). Given that previous studies have shown that Piezo1 channels are inhibited by acid (Bae et al., 2015), these data suggest that the mechanical force- or acid-induced current in Piezo1(1–2190)-ASIC1-transfected cells is mediated by the ASIC channel pore of the chimera protein. (4) The MA currents of the chimera were partially blocked by amiloride at 100 mM concentration that effectively blocked ASIC1, but not Piezo1 (Figures 7E–7H in Zhao et al., 2016), further supporting the idea that the ion-conducting pore is distinct from the Piezo1 channel pore. Notably, the blocking effect of amiloride on the chimeric channel was less efficient than on ASIC1 (49.7% ± 9.0% versus 14.1% ± 5.2%, respectively) (Figures 7E–7H in Zhao et al., 2016), suggesting that attachment of the bulky N-terminal region of mPiezo1 might alter the pore properties of ASIC1. (5) The reversal potential of the chimera was significantly different from that of the sodium-selective ASIC1 and non-selective mPiezo1, indicating that the MA current of the chimeric channel was non-selective for Na+ over K+ despite the presence of the ASIC1 channel pore (Figures S5D–S5F in Zhao et al., 2016). Given that ASIC1 channels are capable of adopting both Na+-selective and non-selective conformations of their ion-conducting pore (i.e., the PcTX1 [a spider toxin]-bound ASIC1 at neutral pH forms a non-selective cation channel, while at low pH is Na+ selective) (Baconguis and Gouaux, 2012), it is not totally unexpected that the bulky N-terminal region of mPiezo1 might cause alteration of the pore properties of the fused ASIC1. We hypothesize that the ion selectivity of the Piezo1 channel pore could be flexible, which merits further investigation. Based on the characterization of the Piezo1(1–2190)-ASIC1 chimera and the structural organization of Piezo1, we suggest that the peripheral propeller structure formed by the N-terminal region of 1–2190 might in principle be sufficient to confer mechanosensitivity to Piezo1 channels. Neuron 94, 274–277, April 19, 2017 275
Evidence Inconsistent with the Suggestion that the Piezo1(1–2190)-ASIC1-Mediated Current Is Due to Endogenous Piezo1 in HEK293T Cells The expression of endogenous Piezo1 in HEK293 cells is too low to even be reliably detected by RT-PCR, which has been clearly pointed out by Dubin et al. in the Matters Arising as well. Paradoxically, they have shown that 11 out of 25 vectortransfected cells were found to have MA currents. It is not clear why the wild-type HEK293T cells in the Matters Arising have such a high percentage of MA currents. In our experience and under the experimental conditions used in the previous study (Zhao et al., 2016), the chance to detect endogenous MA currents in HEK293T cells is rare. For instance, we only detected 2 out of 20 vector-transfected HEK293 cells that had MA currents with an amplitude of about 20 pA. Previous studies by Coste et al. showed negligible MA currents from vector-transfected HEK293 cells as well (Coste et al., 2010). By contrast, the average maximal current of Piezo1(1–2190)ASIC1 is 132.5 ± 24.3 pA (Zhao et al., 2016). One characteristic feature of the endogenous MA currents of wild-type HEK293 cells shown in the Matters Arising is the apparent lack of inactivation (Figure 1 of Dubin et al., 2017). However, the chimera-mediated MA currents display clear inactivation, as shown in Figure 7 of our study (Zhao et al., 2016) as well as in Figures 2 and 3 of Dubin et al. (2017). Dubin et al. have suggested that overexpression of Piezo1(1– 2190)-ASIC1 might potentiate the expression of endogenous Piezo1. However, as described above, the chimera-mediated current is clearly distinct from the wild-type Piezo1-mediated current. Furthermore, the authors in the Matters Arising observed that the apparent threshold for responsive vectortransfected HEK293T cells (12.4 ± 1.1 mm) was about twice that observed for cells expressing the chimera (6 mm). It is striking that the threshold of the chimera is comparable to that of the exogenously expressed Piezo1 in HEK293T cells (5.8 ± 0.8 mm), as shown in the Matters Arising. In our study (Zhao et al., 2016), the thresholds for Piezo1(1–2190)ASIC1 and Piezo1 in HEK293T cells were 8.8 ± 0.90 mm and 5.25 ± 0.52 mm, respectively. Thus, both studies consistently show that the threshold of the chimera-mediated MA currents is clearly distinct from the endogenous MA currents observed in HEK293T cells by Dubin et al. Another possibility suggested by Dubin et al. is that overexpression of the Piezo1(1–2190)-ASIC1 chimera might lead to unknown stresses to cells that indirectly modulate Piezo1 function. However, this appears to be inconsistent with our observation that transfection of other chimeras differing slightly in amino acid compositions, including Piezo1(1–2170)ASIC1, Piezo1(1–2181)-ASIC1, Piezo1(1–2190)-ASIC1 (without the GGGGG linker), and Piezo1(1–2190)-P2X4 (with a GGGGG linker in between), failed to induce MA currents. Alternatively, the overexpressed Piezo1(1–2190)-ASIC1 proteins might form heteromeric channels with endogenous Piezo1. This might explain the altered current properties recorded from the chimera-transfected HEK293T cells. However, in this scenario, as the authors in the Matters Arising pointed out, the transfected construct Piezo1-ASIC1 is expected to be logs in excess of any endogenous Piezo1. The heteromeric channel, if it exists, 276 Neuron 94, 274–277, April 19, 2017
is more likely to be composed of two Piezo1(1–2190)-ASIC1 subunits and one endogenous Piezo1 subunit (assuming that the heteromeric channel also has a trimeric architecture). This would still support the idea that the N-terminal region of Piezo1 might confer mechanosensitivity to the heteromeric pore. Other Scenarios that Might Account for the Lesser Effectiveness of Piezo1(1–2190)-ASIC1 in Mediating MA Currents in the HEK-P1 Knockout Cells Currently, it is not clear why overexpression of the Piezo1(1– 2190)-ASIC1 protein in the HEK-P1 knockout cell line was less effective in generating MA currents than in HEK293T cells. One possibility is that the truncated Piezo1 proteins (1–1984) (without the C-terminal pore-forming domain) might cause dominantnegative suppression of the chimeric proteins if the chimeramediated currents observed in wild-type HEK293 cells are due to formation of heteromeric channels with the endogenous Piezo1 proteins. Another possibility is that the HEK-P1 knockout cell line might not be fully capable of supporting all mechanosensitive ion channels. Indeed, in addition to functioning as ion-permeating pores, mechanosensitive ion channels such as TREK1 have been shown to have a drastic effect on cytoskeleton and cell morphology (Lauritzen et al., 2005). Thus, it is possible that Piezo1 might underlie some roles (not necessary related to its ion-conducting pore function) in affecting some unclear aspects of cellular properties. In line with this idea, Piezo1 has been shown to be able to activate integrin and affect cell migration (McHugh et al., 2010, 2012). Indeed, Dubin et al. have noticed that the HEK-P1 knockout cell line requires much a longer time (up to four to five passages) for recovery after thawing, indicating potential abnormity and adaptation of the cells. It is expected that overexpression of Piezo1 itself in the knockout cell line should fully rescue any phenotypes due to its depletion and thus generate Piezo1-mediated MA currents. However, this does not mean that the knockout cell line might support mechanotransduction of all other mechanosensitive ion channels, including Piezo2, NOMPC, TREK, TMEM150C, and Piezo1(1– 2190)-ASIC1. Furthermore, the method used by the authors in the Matters Arising for generating the Piezo1 knockout cell line is prone to introducing non-specific mutations in the whole genome, which might cause undetected effects on the cellular properties of the cell line. For instance, what causes the observed slower recovery of the HEK-P1 knockout cell line? Thus, the chimera should be tested in different clones of HEK-P1 knockout cell lines as well as in those clones that had experienced the same selection processes during the generation of the knockout cell line, but with the Piezo1 gene remaining intact. In summary, the finding that transfection of HEK293T cells with the Piezo1(1–2190)-ASIC1 chimera can give rise to MA currents has been consistently observed. The new finding by Dubin et al. with the use of the HEK-P1 knockout cells complicates the interpretation of the implication of the chimera results. Nevertheless, together with the structural features of the Piezo1 channel (Ge et al., 2015), the hypothesis that the N-terminal propeller-resembling structure might serve as an intrinsic mechanotransduction module for the Piezo1 channel remains alive for further testing.
For instance, future studies should focus on identifying specific regions/residues in the N-terminal region that critically determine the mechanotransduction process of the Piezo1 channel. For a novel and complicated ion channel family like Piezos, it is reasonable to believe that extensive investigations need to be done in order to obtain a thorough mechanistic understanding, which might eventually allow explanation of any paradox arising during the course of the study. AUTHOR CONTRIBUTIONS B.X. wrote the paper and all authors discussed and read the paper. ACKNOWLEDGMENTS
Coste, B., Mathur, J., Schmidt, M., Earley, T.J., Ranade, S., Petrus, M.J., Dubin, A.E., and Patapoutian, A. (2010). Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330, 55–60. Coste, B., Xiao, B., Santos, J.S., Syeda, R., Grandl, J., Spencer, K.S., Kim, S.E., Schmidt, M., Mathur, J., Dubin, A.E., et al. (2012). Piezo proteins are pore-forming subunits of mechanically activated channels. Nature 483, 176–181. Dubin, A.E., Murthy, S., Lewis, A.H., Brosse, L., Cahalan, S.M., Grandl, J., Coste, B., and Patapoutian, A. (2017). Endogenous Piezo1 can confound mechanically activated channel identification and characterization. Neuron 94, this issue, 266–270. Ge, J., Li, W., Zhao, Q., Li, N., Chen, M., Zhi, P., Li, R., Gao, N., Xiao, B., and Yang, M. (2015). Architecture of the mammalian mechanosensitive Piezo1 channel. Nature 527, 64–69.
This work was supported by the National Natural Science Foundation of China (31422027, 31371118, and 31630090), the Ministry of Science and Technology (2015CB910102 and 2016YFA0500402), and the Ministry of Education (Young Thousand Talent Program) to B.X.
Hong, G.-S., Lee, B., and Oh, U. (2017). Evidence for mechanosensitive channel activity of tentonin 3/TMEM150C. Neuron 94, this issue, 271–273.
Received: December 6, 2016 Revised: January 22, 2017 Accepted: March 27, 2017 Published: April 19, 2017
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