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At its most fundamental level, touch sensation requires the translation of mechanical energy into mechanosensitive ion channel opening, thereby generating electro-chemical signals. Our understanding of this process, especially how the cytoskeleton influences it, remains unknown. Here we demonstrate that mice lacking the α-tubulin acetyltransferase Atat1 in sensory neurons display profound deficits in their ability to detect mechanical stimuli. We show that all cutaneous afferent subtypes, including nociceptors have strongly reduced mechanosensitivity upon Atat1 deletion, and that consequently, mice are largely insensitive to mechanical touch and pain. We establish that this broad loss of mechanosensitivity is dependent upon the acetyltransferase activity of Atat1, which when absent leads to a decrease in cellular elasticity. By mimicking α-tubulin acetylation genetically, we show both cellular rigidity and mechanosensitivity can be restored in Atat1 deficient sensory neurons. Hence, our results indicate that by influencing cellular stiffness, α-tubulin acetylation sets the force required for touch. DOI: http://dx.doi.org/10.7554/eLife.20813.001

Mechanical forces are important for normal gastrointestinal development and function. Now, He et al. have discovered a population of Drosophila midgut epithelial enteroendocrine cell (EEC) precursors that express the mechanosensitive ion channel Piezo, which generates a calcium influx that drives EEC differentiation and proliferation in response to physiological mechanical stimuli.

Immunology In lymph nodes, fibroblastic reticular cells (FRCs) and associated collagen fibers assemble into a network of “conduits.” The network is believed to allow the swift and efficient uptake of lymph-borne antigens. The role of these networks in other lymphoid tissues is less well described. Chang et al. characterized a similar conduit system in Peyer's patches (PPs) in the intestine. In this case, the conduits regulate the flow of absorbed luminal fluid. The maintenance of this system by FRCs depends on luminal flow and is needed to sustain lymphocyte recruitment to PPs and for effective humoral immune responses. Intriguingly, FRCs perceive luminal flow by means of the mechanosensitive ion channel Piezo1. Nat. Immunol. 20 , 1506 (2019).

PIEZO1 is an inactivating eukaryotic cation-selective mechanosensitive ion channel. Two sites have been located in the channel that when individually mutated lead to xerocytotic anemia by slowing inactivation. By introducing mutations at two sites, one associated with xerocytosis and the other artificial, we were able to remove inactivation. The double mutant (DhPIEZO1) has a substitution of arginine for methionine (M2225R) and lysine for arginine (R2456K). The loss of inactivation was accompanied by ∼30-mmHg shift of the activation curve to lower pressures and slower rates of deactivation. The slope sensitivity of gating was the same for wild-type and mutants, indicating that the dimensional changes between the closed and open state are unaffected by the mutations. The unitary channel conductance was unchanged by mutations, so these sites are not associated with pore. DhPIEZO1 was reversibly inhibited by the peptide GsMTx4 that acted as a gating modifier. The channel kinetics were solved using complex stimulus waveforms and the data fit to a three-state loop in detailed balance. The reaction had two pressure-dependent rates, closed to open and inactivated to closed. Pressure sensitivity of the opening rate with no sensitivity of the closing rate means that the energy barrier between them is located near the open state. Mutant cycle analysis of inactivation showed that the two sites interacted strongly, even though they are postulated to be on opposite sides of the membrane.

The search for new and effective antimicrobial agents has never been as important; however, since the discovery of antibiotics, exploring the antimicrobial activity of dyes has been forgotten. Antimicrobial dyes are an untapped resource and have the ability to potentially combat the spread of drug-resistant bacteria either alone or as antimicrobial adjuvants. The mechanosensitive ion channel of large conductance (MscL) is highly conserved and ubiquitous in bacterial species. There is evidence to suggest that at least one triphenylmethane dye acts through the highly conserved MscL channel and combining the two approaches of exploring the mechanism of action of other triphenylmethane dyes or antimicrobial dyes in general and the novel MscL target provides a new opportunity for further exploration.

The phenotypic switch underlying the differentiation of cardiac fibroblasts into hypersecretory myofibroblasts is critical for cardiac remodeling following myocardial infarction. Myofibroblasts facilitate wound repair in the myocardium by secreting and organizing extracellular matrix (ECM) during the wound healing process. However, the molecular mechanisms involved in myofibroblast differentiation are not well known. TGF-β has been shown to promote differentiation and this, combined with the robust mechanical environment in the heart, lead us to hypothesize that the mechanotransduction and TGF-β signaling pathways play active roles in the differentiation of cardiac fibroblasts to myofibroblasts. Here, we show that the mechanosensitve ion channel TRPV4 is required for TGF-β1-induced differentiation of cardiac fibroblasts into myofibroblasts. We found that the TRPV4-specific antagonist AB159908 and siRNA knockdown of TRPV4 significantly inhibited TGFβ1-induced differentiation as measured by incorporation of α-SMA into stress fibers. Further, we found that TGF-β1-induced myofibroblast differentiation was dependent on ECM stiffness, a response that was attenuated by TRPV4 blockade. Finally, TGF-β1 treated fibroblasts exhibited enhanced TRPV4 expression and TRPV4-mediated calcium influx compared to untreated controls. Taken together these results suggest for the first time that the mechanosensitive ion channel, TRPV4, regulates cardiac fibroblast differentiation to myofibroblasts by integrating signals from TGF-β1 and mechanical factors.

All cells respond to osmotic challenges, including those imposed during normal growth and development. Mechanosensitive (MS) ion channels provide a conserved mechanism for regulating osmotic forces by conducting ions in response to increased membrane tension. We previously demonstrated that the MS ion channel MscS-Like 8 (MSL8) is required for pollen to survive multiple osmotic challenges that occur during the normal process of fertilization, and that it can inhibit pollen germination. However, it remained unclear whether these physiological functions required ion flux through a mechanically gated channel provided by MSL8. We introduced two point mutations into the predicted pore-lining domain of MSL8 that disrupted normal channel function in different ways. The Ile711Ser mutation increased the tension threshold of the MSL8 channel while leaving conductance unchanged, and the Phe720Leu mutation severely disrupted the MSL8 channel. Both of these mutations impaired the ability of MSL8 to preserve pollen viability during hydration and to maintain the integrity of the pollen tube when expressed at endogenous levels. When overexpressed in a msl8-4 null background, MSL8I711S could partially rescue loss-of-function phenotypes, while MSL8F720L could not. When overexpressed in the wild type Ler background, MSL8I711S suppressed pollen germination, similar to wild type MSL8. In contrast, MSL8F720L failed to suppress pollen germination and increased pollen bursting, thereby phenocopying the msl8-4 mutant. Thus, an intact MSL8 channel is required to for normal pollen function during hydration and germination. These data establish MSL8 as the first plant MS channel to fulfill previously established criteria for assignment as a mechanotransducer.

The 3-D structure of living organisms is influenced by various physical factors, including gravity, atmospheric pressure, and water pressure. These constant, seemingly unremarkable mechanical forces are in fact essential for forming, maintaining, and changing the dynamic structures and functions of our cells, tissues, and organs. “Mechanobiology” is the study of the molecular cascades and cellular responses that are triggered by mechanical forces. A more comprehensive and improved understanding of mechanobiology may greatly facilitate the development of new therapies that act by controlling mechanical forces and thereby specifically induce desired molecular, cellular, tissue, and/or organ formation, changes, or repair. We have denoted these therapies as “mechanotherapies.”

During nervous system development, neurons extend axons along well-defined pathways. The current understanding of axon pathfinding is based mainly on chemical signaling. However, growing neurons interact not only chemically but also mechanically with their environment. Here we identify mechanical signals as important regulators of axon pathfinding. In vitro, substrate stiffness determined growth patterns of Xenopus retinal ganglion cell axons. In vivo atomic force microscopy revealed a noticeable pattern of stiffness gradients in the embryonic brain. Retinal ganglion cell axons grew toward softer tissue, which was reproduced in vitro in the absence of chemical gradients. To test the importance of mechanical signals for axon growth in vivo, we altered brain stiffness, blocked mechanotransduction pharmacologically and knocked down the mechanosensitive ion channel piezo1. All treatments resulted in aberrant axonal growth and pathfinding errors, suggesting that local tissue stiffness, read out by mechanosensitive ion channels, is critically involved in instructing neuronal growth in vivo.

Piezo1 is a eukaryotic cation-selective mechanosensitive ion channel. To understand channel function in vivo, we first need to analyze and compare the response in the whole cell and the patch. In patches, Piezo1 inactivates and the current is fit well by a 3-state model with a single pressure-dependent rate. However, repeated stimulation led to an irreversible loss of inactivation. Remarkably, the loss of inactivation did not occur on a channel-by-channel basis but on all channels at the same time. Thus, the channels are in common mechanical domain. Divalent ions decreased the unitary conductance from ~68 pS to ~37 pS, irrespective of the cation species. Mg and Ca did not affect inactivation rates, but Zn caused a 3-fold slowing. CytochalasinD (cytoD) does not alter inactivation rates or the transition to the non-inactivating mode but does reduce the steady-state response. Whole-cell currents were similar to patch currents but also had significant differences. In contrast to the patch, cytoD inhibited the current suggesting that the activating forces were transmitted through the actin cytoskeleton. Hypotonic swelling that prestressed the cytoskeleton and the bilayer greatly increased the sensitivity of both control and cytoD cells so there are two pathways to transmit force to the channels. In contrast to patch, removing divalent ions decreased the whole-cell current. The difference between whole cell and patch properties provide new insights into our understanding of the Piezo1 gating mechanisms and cautions against generalization to in situ behavior.

It has been suggested that mechanosensitive ion channels initiate myogenic responses in vessels; however, the molecular identity of the mechanosensitive ion channel complex is unknown. Although previous reports have suggested that epithelial Na(+) channel (ENaC) proteins are mechanotransducers in arteries, experimental evidence demonstrating that ENaC proteins are mechanotransducers are not fully elucidated. The goal of the present study was to determine whether the ENaC is a mechanotransducer for the myogenic response by providing supporting evidence in the rat posterior cerebral artery (PCA). We measured the effect of ENaC inhibition on the pressure-induced myogenic response, Ca(2+) concentration and 20 kDa myosin light chain (MLC(20)) phosphorylation. We detected expression of βENaC and γENaC subunits in rat PCA by Western blots and immunofluorescence. Inhibition of ENaCs with amiloride, ethyl isopropyl amiloride or benzamil blocked the myogenic response. Moreover, the myogenic response was inhibited in rat PCA transfected with βENaC and γENaC small interfering RNA. The myogenic response was inhibited by elimination of external Na(+), which was replaced with N-methyl-d-glucamine. Amiloride and nifedipine inhibited the pressure-induced increase in Ca(2+) concentration. Finally, MLC(20) increased when the intraluminal pressure was raised, and the pressure-induced increase in MLC(20) phosphorylation was inhibited by pretreatment with amiloride, and in arteries transfected with βENaC or γENaC small interfering RNA. Our results suggest that ENaCs may play an important role as mechanosensitive ion channels initiating pressure-induced myogenic responses in rat PCA.

To gain insight into the cellular functions of the mid1-complementing activity (MCA) family proteins, encoding putative Ca2+-permeable mechanosensitive channels, we isolated two MCA homologs of tobacco (Nicotiana tabacum) BY-2 cells, named NtMCA1 and NtMCA2. NtMCA1 and NtMCA2 partially complemented the lethality and Ca2+ uptake defects of yeast mutants lacking mechanosensitive Ca2+ channel components. Furthermore, in yeast cells overexpressing NtMCA1 and NtMCA2, the hypo-osmotic shock-induced Ca2+ influx was enhanced. Overexpression of NtMCA1 or NtMCA2 in BY-2 cells enhanced Ca2+ uptake, and significantly alleviated growth inhibition under Ca2+ limitation. NtMCA1-overexpressing BY-2 cells showed higher sensitivity to hypo-osmotic shock than control cells, and induced the expression of the touch-inducible gene, NtERF4. We found that both NtMCA1-GFP and NtMCA2-GFP were localized at the plasma membrane and its interface with the cell wall, Hechtian strands, and at the cell plate and perinuclear vesicles of dividing cells. NtMCA2 transcript levels fluctuated during the cell cycle and were highest at the G1 phase. These results suggest that NtMCA1 and NtMCA2 play roles in Ca2+-dependent cell proliferation and mechanical stress-induced gene expression in BY-2 cells, by regulating the Ca2+ influx through the plasma membrane.

Cells can respond to mechanical stress by gating mechanosensitive ion channels (MSCs). The cloning of Piezo1, a eukaryotic cation selective MSC, defines a new system for studying mechanical transduction at the cellular level. Because Piezo1 has electrophysiological properties similar to those of endogenous cationic MSCs that are selectively inhibited by the peptide GsMTx4, we tested whether the peptide targets Piezo1 activity. Extracellular GsMTx4 at micromolar concentrations reversibly inhibited ∼80% of the mechanically induced current of outside-out patches from transfected HEK293 cells. The inhibition was voltage insensitive, and as seen with endogenous MSCs, the mirror image d enantiomer inhibited like the l. The rate constants for binding and unbinding based on Piezo1 current kinetics provided association and dissociation rates of 7.0 × 10(5) M(-1) s(-1) and 0.11 s(-1), respectively, and a K(D) of ∼155 nM, similar to values previously reported for endogenous MSCs. Consistent with predicted gating modifier behavior, GsMTx4 produced an ∼30 mmHg rightward shift in the pressure-gating curve and was active on closed channels. In contrast, streptomycin, a nonspecific inhibitor of cationic MSCs, showed the use-dependent inhibition characteristic of open channel block. The peptide did not block currents of the mechanical channel TREK-1 on outside-out patches. Whole-cell Piezo1 currents were also reversibly inhibited by GsMTx4, and although the off rate was nearly identical to that of outside-out patches, differences were observed for the on rate. The ability of GsMTx4 to target the mechanosensitivity of Piezo1 supports the use of this channel in high-throughput screens for pharmacological agents and diagnostic assays.

The Piezo2 protein senses changes in lung volume, acting in different neurons to convey this information to the brain. This finding adds to the list of roles for Piezo2 in mechanosensation. See Article p.176 The Hering–Breuer inflation reflex, described some 150 years ago, is thought to protect the lung from overinflation thanks to stretch-activated sensory neurons that innervate the lung, but the actual molecular and cellular mechanisms involved have remained unknown. Ardem Patapoutian and colleagues find that this reflex is absent in adult mice that lack the mechanosensitive ion channel Piezo2, which was previously implicated in the skin's sense of touch. Surprisingly, Piezo2 is also required for initial lung inflation at birth, thus establishing a role for mechanotransduction in respiratory control in both newborn and adult mice.

Piezo1 is a mechanosensitive ion channel supposed to regulate the volume and maintain the structural integrity in Red Blood Cells (RBCs), as gain-of-function mutations in this channel are associated to the RBC disease Hereditary Xerocytosis (Zarychanski et al. Blood 2012; Bae et al. Proceedings of the National Academy of Sciences 2013). Piezo1 is activated by several mechanical forces, including stretching, poking and shear stress and allows Ca2+ and other cations to enter the cell generating an electrical response. In 2015, it has been discovered that Piezo1 is sensitive to a small molecule, Yoda1 (Syeda et al. Elife 2015), which keeps the channel open and affects its inactivation kinetics. This finding has created new possibilities to elucidate Piezo1 gating mechanism and explore its functional significance in physiological and pathophysiological conditions. Here, we present a patient with a novel PIEZO1 mutation (R2110W) and a patch clamp-based high-throughput screening assay for Piezo1 activity. We established a protocol to detect functional Piezo1 mutations upon chemical stimulation by Yoda1, yet were not able to stimulate the channel via mechanical force, i.e. pressure steps and shear-stress. The assay was first developed on Neuro2A (N2A), a neuroblastoma cell-line endogenously expressing Piezo1 channels (kindly provided by Max-Delbrück Center, Berlin), due to larger abundance of Piezo1 channels in these cells. Initial experiments were performed on the Patchliner (Nanion Technologies GmbH, Munich), a medium-throughput automated patch clamp system able to record up to 8 cells at a time. Currents were elicited using a voltage ramp ranging from -100 to +80 mV for 300 ms, the holding potential was set to -60 mV. A significantly increased whole-cell current was observed upon 10 µM Yoda1 application in half of the recorded cells and the resulting Yoda1-induced currents were inhibited by 30 µM gadolinium chloride, a non-specific blocker of stretch-activated channels. The assay was then implemented on the SyncroPatch 384PE (Nanion Technologies GmbH, Munich), capable of recording up to 384 cells in parallel under identical experimental conditions, thus allowing for reliable statistical analysis. Yoda1 responding cells were selected based on strict quality control (QC) criteria, i.e. the seal resistance stability over time. In one example NPC-384 chip 140 out of 384 N2A cells (37%) passed the QC criteria and 85 cells (60% of the valid cells) were considered as Yoda1 responders. Finally, we investigated Piezo1 electrophysiological properties in healthy and patient RBCs carrying the novel PIEZO1 R2110W mutation. Similar to N2A cells, RBCs currents were analyzed and divided into Yoda1 responders and non-responders according to our QC criteria. The increase in whole-cell currents induced by Yoda1 application was significantly higher in patient compared to control RBCs, which was also reflected by a higher number of Yoda1 responders compared to control. Residue R2110W is structurally located in a gating sensitive area of the channel protein suggesting a gain-of-function. This would be in line with previously described mutations in PIEZO1 (Albuisson et al. Nature Communications 2013) and the mild form of anaemia observed in the patient. Furthermore, we excluded any involvement of Gardos channels in the Yoda1-induced currents by comparing measurements in the presence and absence of the specific Gardos channel inhibitor TRAM-34. Altogether, our work demonstrates that high-throughput patch clamping can provide a robust assay to study functional Piezo1 impairments in primary RBCs without expressing the mutated channel protein in a heterologous expression system. Our approach may be used to detect other channelopathies not only in RBCs and may serve as routine screening assay for diseases related to ion channel dysfunctions in general, complementary to gene sequencing. Disclosures No relevant conflicts of interest to declare.

Xenon is frequently used as a general anesthetic in humans, but the mechanism remains an issue of debate. While for some membrane proteins, a direct interaction of xenon with the protein has been shown to be the inhibitory mechanism, other membrane protein functions could be affected by changes of membrane properties due to partitioning of the gas into the lipid bilayer. Here, the effect of xenon on a mechanosensitive ion channel and a copper ion-translocating ATPase was compared under different conditions. Xenon inhibited spontaneous gating of the Escherichia coli mechano-sensitive mutant channel MscL-G22E, as shown by patch-clamp recording techniques. Under high hydrostatic pressure, MscL-inhibition was reversed. Similarly, the activity of the Enterococcus hirae CopB copper ATPase, reconstituted into proteoliposomes, was inhibited by xenon. However, the CopB ATPase activity was also inhibited by xenon when CopB was in a solubilized state. These findings suggest that xenon acts by directly interacting with these proteins, rather than via indirect effects by altering membrane properties. Also, inhibition of copper transport may be a novel effect of xenon that contributes to anesthesia.

“Over the past decade, increasing numbers of ion channel crystal structures have been solved, and many more structures will likely be determined in the coming years. Computational modeling can play an important role in verifying and helping to understand these channel crystal structures. This article focuses on a few important ways in which computer modeling has promoted the understanding of bacterial mechanosensitive ion channel crystal structures. Considering these studies of largeand small-conductance mechanosensitive ion channels provides general insights into the role computer modeling can play in elucidating aspects of channel structures that are essential in drug-design efforts.”

Due to the limited self-repair capacity of articular cartilage, the surgical restoration of defective cartilage remains a major clinical challenge. The cell-based approach, which is known as autologous chondrocyte transplantation (ACT), has limited success, presumably because the chondrocytes acquire a fibroblast-like phenotype inmonolayer culture. This unwanted dedifferentiation process is typically addressed by using three-dimensional scaffolds, pellet culture, and/or the application of exogenous factors. Alternative mechanical unloading approaches are suggested to be beneficial in preserving the chondrocyte phenotype. In this study, we examined if the random positioning machine (RPM) could be used to expand chondrocytes in vitro such that they maintain their phenotype. Bovine chondrocytes were exposed to (a) eight days instatic monolayer culture; (b) two days in static monolayer culture, followed by six days of RPMexposure; and, (c) eight days of RPMexposure. Furthermore, the experiment was also conducted with the application of 20 mM gadolinium, which is a nonspecific ion-channel blocker. The results revealed that the chondrocyte phenotype is preserved when chondrocytes go into suspension and aggregate to cell clusters. Exposure to RPM rotation alone does not preserve the chondrocyte phenotype. Interestingly, the gene expression (mRNA) of the mechanosensitive ion channel TRPV4 decreased with progressing dedifferentiation. In contrast, the gene expression (mRNA) of the mechanosensitive ion channel TRPC1 was reduced around fivefold to 10-fold in all of the conditions.The application of gadolinium had only a minor influence on the results. This and previous studies suggest that the chondrocyte phenotype is preserved if cells maintain a round morphology and that the ion channel TRPV4 could play a key role in the dedifferentiation process.

Recent studies suggest certain epithelial Na+channel (ENaC) proteins may be components of mechanosensitive ion channel complexes in vascular smooth muscle cells that contribute to pressure-induced constriction in middle cerebral arteries (MCA). However, the role of a specific ENaC protein, βENaC, in pressure-induced constriction of MCAs has not been determined. The goal of this study was to determine whether pressure-induced constriction in the MCA is altered in a mouse model with reduced levels of βENaC. Using quantitative immunofluorescence, we found whole cell βENaC labeling in cerebral vascular smooth muscle cells (VSMCs) was suppressed 46% in βENaC homozygous mutant (m/m) mice compared with wild-type littermates (+/+). MCAs from βENaC +/+ and m/m mice were isolated and placed in a vessel chamber for myographic analysis. Arteries from βENaC+/+ mice constricted to stepwise increases in perfusion pressure and developed maximal tone of 10 ± 2% at 90 mmHg ( n = 5). In contrast, MCAs from βENaC m/m mice developed significantly less tone (4 ± 1% at 90 mmHg, n = 5). Vasoconstrictor responses to KCl (4–80 mM) were identical between genotypes and responses to phenylephrine (10−7-10−4M) were marginally altered, suggesting that reduced levels of VSMC βENaC specifically inhibit pressure-induced constriction. Our findings indicate βENaC is required for normal pressure-induced constriction in the MCA and provide further support for the hypothesis that βENaC proteins are components of a mechanosensor in VSMCs.

Macrophage phagocytosis of particles and pathogens is an essential aspect of innate host defense. Phagocytic function requires cytoskeletal rearrangements that depend on the interaction between macrophage surface receptors, particulates/pathogens, and the extracellular matrix. In the present study we determine the role of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), in integrating the LPS and matrix stiffness signals to control macrophage phenotypic change for host defense and resolution from lung injury. We demonstrate that active TRPV4 mediates LPS-stimulated murine macrophage phagocytosis of nonopsonized particles (Escherichia coli) in vitro and opsonized particles (IgG-coated latex beads) in vitro and in vivo in intact mice. Intriguingly, matrix stiffness in the range seen in inflamed or fibrotic lung is required to sensitize the TRPV4 channel to mediate the LPS-induced increment in macrophage phagocytosis. Furthermore, TRPV4 is required for the LPS induction of anti-inflammatory/proresolution cytokines. These findings suggest that signaling through TRPV4, triggered by changes in extracellular matrix stiffness, cooperates with LPS-induced signals to mediate macrophage phagocytic function and lung injury resolution. These mechanisms are likely to be important in regulating macrophage function in the context of pulmonary infection and fibrosis.

Piezo ion channels are activated by various types of mechanical stimuli and function as biological pressure sensors in both vertebrates and invertebrates. To date, mechanical stimuli are the only means to activate Piezo ion channels and whether other modes of activation exist is not known. In this study, we screened ∼3.25 million compounds using a cell-based fluorescence assay and identified a synthetic small molecule we termed Yoda1 that acts as an agonist for both human and mouse Piezo1. Functional studies in cells revealed that Yoda1 affects the sensitivity and the inactivation kinetics of mechanically induced responses. Characterization of Yoda1 in artificial droplet lipid bilayers showed that Yoda1 activates purified Piezo1 channels in the absence of other cellular components. Our studies demonstrate that Piezo1 is amenable to chemical activation and raise the possibility that endogenous Piezo1 agonists might exist. Yoda1 will serve as a key tool compound to study Piezo1 regulation and function. DOI: http://dx.doi.org/10.7554/eLife.07369.001, eLife digest Within our bodies, cells and tissues are constantly being pushed and pulled by their surrounding environment. These mechanical forces are then transformed into electrical or chemical signals by cells. This process is crucial for many biological structures, such as blood vessels, to develop correctly, and is also a key part of our senses of touch and hearing. In 2010, researchers discovered a group of ion channels—proteins embedded in the membrane that surrounds a cell—that open up when a force is applied and allow ions such as calcium, potassium, and sodium to flow. This movement of ions generates the electrical response of the cell to the applied force. However, not much is known about how these ‘Piezo’ ion channels work. To investigate this, it is important to be able to precisely control how and when the Piezo channels open. Many other ion channels are studied by using small chemical compounds to activate them, but there were none that were known to act on Piezo proteins. Syeda et al.—including some of the researchers involved in the 2010 work—screened over three million compounds for their ability to cause calcium ions to flow into human cells to try to identify chemicals that activate the Piezo channels. This revealed one promising candidate named Yoda1, which specifically activated Piezo1: a Piezo protein that had previously been linked to a role in blood vessel development in embryos. To investigate how Yoda1 activates Piezo1, Syeda et al. placed Piezo1 in an artificial cell membrane that did not contain any other cellular components. When Yoda1 was added to this set up, the Piezo1 channels opened up. This suggests that Piezo1 and Yoda1 interact in a manner that does not require additional cellular components other than a cell membrane. Separate work by Cahalan, Lukacs et al. uses Yoda1 to reveal that Piezo1 helps to control the volume of red blood cells, showing that in the future, Yoda1 could be valuable in research that investigates the roles of Piezo1. DOI: http://dx.doi.org/10.7554/eLife.07369.002

Calcium plays an essential role in a variety of stress responses of eukaryotic cells; however, its function in prokaryotes is obscure. Bacterial ion channels that transport Ca(2+) are barely known. We investigated temperature-induced changes in intracellular concentration of Ca(2+), Na(+) and K(+) in the cyanobacterium Synechocystis sp. strain PCC 6803 and its mutant that is defective in mechanosensitive ion channel MscL. Concentration of cations rapidly and transiently increased in wild-type cells in response to cold and heat treatments. These changes in ionic concentrations correlated with the changes in cytoplasmic volume that transiently decreased in response to temperature treatments. However, no increase in ionic concentrations was observed in the MscL-mutant cells. It implies that MscL functions as a non-specific ion channel, and it participates in regulation of cell volume under temperature-stress conditions.

Red blood cells (RBCs) experience significant mechanical forces while recirculating, but the consequences of these forces are not fully understood. Recent work has shown that gain-of-function mutations in mechanically activated Piezo1 cation channels are associated with the dehydrating RBC disease xerocytosis, implicating a role of mechanotransduction in RBC volume regulation. However, the mechanisms by which these mutations result in RBC dehydration are unknown. In this study, we show that RBCs exhibit robust calcium entry in response to mechanical stretch and that this entry is dependent on Piezo1 expression. Furthermore, RBCs from blood-cell-specific Piezo1 conditional knockout mice are overhydrated and exhibit increased fragility both in vitro and in vivo. Finally, we show that Yoda1, a chemical activator of Piezo1, causes calcium influx and subsequent dehydration of RBCs via downstream activation of the KCa3.1 Gardos channel, directly implicating Piezo1 signaling in RBC volume control. Therefore, mechanically activated Piezo1 plays an essential role in RBC volume homeostasis. DOI: http://dx.doi.org/10.7554/eLife.07370.001, eLife digest Within our bodies, cells and tissues are constantly being pushed and pulled by their surrounding environment. These mechanical forces are then transformed into electrical or chemical signals by cells. This process is crucial for many biological structures, such as blood vessels, to develop correctly, and is also a key part of our senses of touch and hearing. In 2010, researchers discovered a group of ion channels—proteins embedded in the membrane that surrounds a cell—that open up when a force is applied and allow calcium and other ions to enter the cell. This movement of ions generates the electrical response of the cell to the applied force. However, not much is known about the roles of these ‘Piezo’ ion channels. Red blood cells experience significant forces when they pass through narrow blood vessels. In a disease called xerocytosis, the red blood cells become severely dehydrated and shrink. In 2013, researchers discovered that patients with this disease have mutations in the gene that codes for the Piezo1 protein: a Piezo protein that has also been linked to a role in blood vessel development in embryos. This suggested that Piezo1 may regulate the volume of red blood cells. Cahalan, Lukacs et al.—including some of the researchers who worked on the 2010 and 2013 studies—have now investigated the role of Piezo1 in red blood cells in more detail. Applying strong forces to red blood cells from mice caused calcium to rapidly enter cells through Piezo1 channels. Cahalan, Lukacs et al. then deleted the Piezo1 gene from red blood cells. This made the cells larger and more fragile than normal cells because they contained too much water. To investigate how Piezo1 regulates water content, the cells were treated with a chemical compound called Yoda1. This compound was shown in a separate study by Syeda et al. to activate Piezo1 channels. Activating Piezo1 caused a second type of ion channel to open up as well, which allowed potassium ions and water molecules to leave the cell. This resulted in the cell becoming dehydrated. This work raises the possibility that Piezo proteins are involved in other diseases where red blood cell volume is altered. In particular, many believe that Piezo1 may be involved in sickle cell disease, a possibility that can now be tested using the tools described in this study. DOI: http://dx.doi.org/10.7554/eLife.07370.002

Immobilisation of cells is an important feature of many cellular assays, as it enables the physical/chemical stimulation of cells; whilst, monitoring cellular processes using microscopic techniques. Current approaches for immobilising cells, however, are hampered by time-consuming processes, the need for specific antibodies or coatings and adverse effects on cell integrity. Here, we present a dielectrophoresis-based approach for the robust immobilisation of cells and analysis of their responses under high shear flows. This approach is quick and label-free and more importantly, minimises the adverse effects of electric field on the cell integrity, by activating the field for a short duration of 120 s, just long enough to immobilise the cells, after which cell culture media (such as HEPES) is flushed through the platform. In optimal conditions, at least 90% of the cells remained stably immobilised, when exposed to a shear stress of 63 dyn/cm2. This approach was used to examine the shear-induced calcium signalling of HEK-293 cells expressing a mechanosensitive ion channel, transient receptor potential vaniloid type 4 (TRPV4), when exposed to the full physiological range of shear stress.

Since the first discovery of mechanosensitive ion channel (MSC) in non-sensory cells in 1984, a variety of MSCs has been identified both in prokaryotic and eukaryotic cells. One of the central issues concerning MSCs is to understand the molecular and biophysical mechanisms of how mechanical forces activate/open MSCs. It has been well established that prokaryotic (mostly bacterial) MSCs are activated exclusively by membrane tension. Thus the problem to be solved with prokaryotic MSCs is the mechanisms how the MSC proteins receive tensile forces from the lipid bilayer and utilize them for channel opening. On the other hand, the activation of many eukaryotic MSCs crucially depends on tension in the actin cytoskeleton. By using the actin cytoskeleton as a force sensing antenna, eukaryotic MSCs have obtained sophisticated functions such as remote force sensing and force-direction sensing, which bacterial MSCs do not have. Actin cytoskeletons also give eukaryotic MSCs an interesting and important function called “active touch sensing”, by which cells can sense rigidity of their substrates. The contractile actin cytoskeleton stress fiber (SF) anchors its each end to a focal adhesion (FA) and pulls the substrate to generate substrate-rigidity-dependent stresses in the FA. It has been found that those stresses are sensed by some Ca2+-permeable MSCs existing in the vicinity of FAs, thus the MSCs work as a substrate rigidity sensor that can transduce the rigidity into intracellular Ca2+ levels. This short review, roughly constituting of two parts, deals with molecular and biophysical mechanisms underlying the MSC activation process mostly based on our recent studies; (1) structure-function in bacterial MSCs activation at the atomic level, and (2) roles of actin cytoskeletons in the activation of eukaryotic MSCs.

The oocytes of the African clawed frog (Xenopus laevis) comprise one of the most widely used membrane protein expression systems. While frequently used for studies of transporters and ion channels, the application of this system to the study of mechanosensitive ion channels has been overlooked, perhaps due to a relative abundance of native expression systems. Recent advances, however, have illustrated the advantages of the oocyte system for studying plant and bacterial mechanosensitive channels. Here we describe in detail the methods used for heterologous expression and characterization of bacterial and plant mechanosensitive channels in Xenopus oocytes.

We suggest that the crystal structure of the mechanosensitive channel of small conductance is in a minimally conductive state rather than being fully activated. Performing Brownian dynamics simulations on the crystal structure show that no ions pass through it. When simulations are conducted on just the transmembrane domain (excluding the cytoplasmic residues 128 to 280) ions are seen to pass through the channel, but the conductance of approximately 30 pS is well below experimentally measured values. The mutation L109S that replaces a pore lining hydrophobic residue with a polar one is found to have little effect on the conductance of the channel. Widening the hydrophobic region of the pore by 2.5 Angstrom however, increases the channel conductance to over 200 pS suggesting that only a minimal conformational change is required to gate the pore.

Open channels: Chemical modulators are developed to convert a naturally occurring channel protein into a pH-actuated nanovalve. The pH interval, sensitivity, and activation of the valve by channel opening are tunable through the design of the modulators. The valve is useful for releasing or mixing the contents of liposomes at a desired location, time, and dosage, for example, in micro/nanosensory and delivery devices (see picture).

Many physiological processes such as cell division, endocytosis and exocytosis cause severe local curvature of the cell membrane. Local curvature has been shown experimentally to modulate numerous mechanosensitive (MS) ion channels. In order to quantify the effects of local curvature we introduced a coarse grain representative volume element for the bacterial mechanosensitive ion channel of large conductance (MscL) using continuum elasticity. Our model is designed to be consistent with the channel conformation in the closed and open states to capture its major continuum rheological behavior in response to the local membrane curvature. Herein we show that change in the local curvature of the lipid bilayer can modulate MscL activity considerably by changing both bilayer thickness and lateral pressure profile. Intriguingly, although bending in any direction results in almost the same free-energy cost, inward (cytoplasmic) bending favors channel opening, whereas outward (periplasmic) bending facilitates closing of the narrowest part of the MscL pore. This quantitative study using MscL as a model channel may have wide reaching consequences for the effect of local curvature on the physiological function of other types of prokaryotic and eukaryotic membrane proteins.

The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals. It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive. Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell-neurite complexes. It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron; however, major aspects of touch sensation remain intact without Merkel cell activity. Here we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, we find that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation.

Characean internodal cells generate receptor potential in response to mechanical stimuli. We studied responses of internodal cells to a long-lasting stimulus and the results were as follows. (i) The cell generated receptor potential at the moment of both compression and decompression. (ii) The receptor potential (DeltaE (m)) was significantly larger at the moment of decompression than at compression. (iii) The longer the duration of the stimulus, the larger was the magnitude of DeltaE (m) at the moment of decompression. (iv) Aequorin studies revealed that the increase in [Ca(2+)](c) (Delta[Ca(2+)](c)) took place at the moment of both compression and decompression. (v) The amplitude of Delta[Ca(2+)](c) was larger at the moment of decompression than at compression, as was the case for DeltaE (m). It was suggested that the amplitude of the receptor potential had a tight correlation with the degree of membrane deformation. We discussed the ionic mechanism of mechano-perception under a long-lasting stimulus in relation to mechanosensitive activation of Ca(2+) channels at the plasma membrane.

Toward the realization of nanoscale device control, we report a molecular valve embedded in a membrane that can be opened by illumination with long-wavelength ultraviolet (366 nanometers) light and then resealed by visible irradiation. The valve consists of a channel protein, the mechanosensitive channel of large conductance (MscL) from Escherichia coli , modified by attachment of synthetic compounds that undergo light-induced charge separation to reversibly open and close a 3-nanometer pore. The system is compatible with a classical encapsulation system, the liposome, and external photochemical control over transport through the channel is achieved.

MscL is a mechanosensitive channel gated by membrane tension in the lipid bilayer alone. Its structure, known from x-ray crystallography, indicates that it is a homopentamer. Each subunit comprises two transmembrane segments TM1 and TM2 connected by a periplasmic loop. The closed pore is lined by five TM1 helices. We expressed in Escherichia coli and purified two halves of the protein, each containing one of the transmembrane segments. Their electrophysiological activity was studied by the patch-clamp recording upon reconstitution in artificial liposomes. The TM2 moiety had no electrophysiological activity, whereas the TM1 half formed channels, which were not affected by membrane tension and varied in conductance between 50 and 350 pS in 100mM KCl. Coreconstitution of the two halves of MscL however, yielded mechanosensitive channels having the same conductance as the native MscL (1500 pS), but exhibiting increased sensitivity to pressure. Our results confirm the current view on the functional role of TM1 and TM2 helices in the MscL gating and emphasize the importance of helix-helix interactions for the assembly and functional properties of the channel protein. In addition, the results indicate a crucial role of the periplasmic loop for the channel mechanosensitivity.

Cells of the cyanobacteriumSynechocystissp. PCC 6803 are equipped with a mechanosensitive ion channel MscL that is located in their plasma membrane. However, the exact function of the channel in this freshwater cyanobacterium is unknown. This study shows that cells ofSynechocystisare capable of releasing Ca2+in response to depolarization of the plasma membrane by the K+ionophore valinomycin in the presence of K+or by tetraphenylphosphonium (TPP+). A fluorescent dye, diS-C3-(5), sensitive to membrane potential and the metallochromic Ca2+indicator arsenazo III were used to follow the plasma membrane depolarization and the Ca2+release, respectively. The Ca2+release from wild-type cells was temperature-dependent and it was strongly inhibited by the Ca2+channel blocker verapamil and by the mechanosensitive channel blocker amiloride. In MscL-deficient cells, Ca2+release was about 50 % of that from the wild-type cells. The mutant cells had lost temperature sensitivity of Ca2+release completely. However, verapamil and amiloride inhibited Ca2+release from these cells in same manner as in the wild-type cells. This suggests the existence of additional Ca2+transporters inSynechocystis, probably of a mechanosensitive nature. Evidence for the putative presence of intracellular Ca2+stores in the cells was obtained by following the increase in fluorescence intensity of the Ca2+indicator chlortetracycline. These results suggest that the MscL ofSynechocystismight operate as a verapamil/amiloride-sensitive outward Ca2+channel that is involved in the plasma-membrane depolarization-induced Ca2+release from the cells under temperature stress conditions.

Mechanosensitive ion channels are gated open and shut by changes in membrane tension enabling the conversion of mechanical stimuli into changes in ionic composition and electrical signalling. However, the precise structural and biophysical mechanisms underlying these processes remain unclear. TREK-2 is a eukaryotic mechanosensitive ion channel that belongs to the Two-Pore Domain (K2P) family of K+ channels and is widely expressed in both the central and peripheral nervous system. I will discuss our recent structural, functional and computational studies which examine how TREK-2 responds to stretch-induced changes in the large lateral forces that vary with depth across the bilayer, and in particular how the asymmetric structure of the channel contributes to this process.

PIEZO1 is a eukaryotic mechanosensitive ion channel that is cation selective and rapidly inactivates. Using fluid shear stress as a stimulus generated in a microfluidic chamber, we have monitored PIEZO activity in the presence of various peptides. To do so, we first developed a cell line that overexpresses the PIEZO1 channel in HEK293T cells. We estimate that there are about 5000 channels per cell. These cells are seeded in the microfluidic chamber and loaded with calcium indicator. A pressure servo is fitted to the chamber and allows control of the applied shear stress stimulus. The calcium change is monitored as an increase in fluorescence. Applying a 10 ms pulse at 13 dyn/cm2, we observed a robust response for the PIEZO1 cell line well above the background observed for HEK293T cells only. Peak response is achieved within a few seconds and is followed by adaptation. Disrupters of the cytoskeleton applied to the cell line, such as cytochalasinD and cholchicine, rendered the PIEZO1 channel unresponsive to the stimulus. The peptide GsMTx4 inhibits PIEZO1 currents. We measured the effect of GsMTx4 on PIEZO1 activity as a way to compare the microfluidic approach to previous work. We applied various concentrations and measure the peak response. The inhibition curve showed a Ki of around 250 nM similar to the value measured previously by patch clamp. Next, we examined the effect of Abeta peptides on PIEZO1 activity. A known attribute of these peptides is their ability to bind membranes. Given PIEZO1 is modulated by membrane tension we wanted to determine how the channel behaved in the presence of Abeta peptides. We prepared the monomer form of the peptide and measured the peak response as a function of peptide concentration. At the same time we prepared the D form of these peptides and measured them as well. Our data show that both the L and D form of Abeta 1-40 and 1-42 inhibit channel function in the fM to pM range. The enantiomeric forms of each peptide were similar in their ability to inhibit PIEZO1 activity. The lack of stereospecific interaction indicates that these peptides modulate activity of the channel by changing the properties of the membrane.This work was funded by NIHLBI and NINDS.

The human mechanosensitive ion channel Piezo1 gates in response to membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently little is known regarding theplasma membrane localization and organization of Piezo1, but previous work suggests that membrane cholesterol content is a key determinant of Piezo1 function. Using a previously characterised Piezo1-GFP fusion protein (hP1-1591-GFP[1]), we investigated the effect of the cholesterol depleting agent methyl-β-Cyclodextrin (mβCD) on the membrane organization and the response of Piezo1 to mechanical force in HEK-293 cells.

Podocytes are specialized, highly differentiated epithelial cells in the kidney glomerulus that are exposed to glomerular capillary pressure and possible increases in mechanical load. The proteins sensing mechanical forces in podocytes are unconfirmed, but the classic transient receptor potential channel 6 (TRPC6) interacting with the MEC-2 homolog podocin may form a mechanosensitive ion channel complex in podocytes. Here, we observed that podocytes respond to mechanical stimulation with increased intracellular calcium concentrations and increased inward cation currents. However, TRPC6-deficient podocytes responded in a manner similar to that of control podocytes, and mechanically induced currents were unaffected by genetic inactivation of TRPC1/3/6 or administration of the broad-range TRPC blocker SKF-96365. Instead, mechanically induced currents were significantly decreased by the specific P2X purinoceptor 4 (P2X4) blocker 5-BDBD. Moreover, mechanical P2X4 channel activation depended on cholesterol and podocin and was inhibited by stabilization of the actin cytoskeleton. Because P2X4 channels are not intrinsically mechanosensitive, we investigated whether podocytes release ATP upon mechanical stimulation using a fluorometric approach. Indeed, mechanically induced ATP release from podocytes was observed. Furthermore, 5-BDBD attenuated mechanically induced reorganization of the actin cytoskeleton. Altogether, our findings reveal a TRPC channel-independent role of P2X4 channels as mechanotransducers in podocytes.

The gating behaviour of a single ion channel can be described by hidden Markov models (HMMs), forming the basis for statistical analysis of patch clamp data. Extensive improved bandwidth (25 kHz, 50 kHz) data from the mechanosensitive channel of large conductance in Escherichia coli were analysed using HMMs, and HMMs with a moving average adjustment for filtering. The aim was to determine the number of levels, and mean current, mean dwell time and proportion of time at each level. Parameter estimates for HMMs with a moving average adjustment for low-pass filtering were obtained using an expectation-maximisation algorithm that depends on a generalisation of Baum’s forward–backward algorithm. This results in a simpler algorithm than those based on meta-states and a much smaller parameter space; hence, the computational load is substantially reduced. In addition, this algorithm maximises the actual log-likelihood rather than that for a related meta-state process. Comprehensive data analyses and comparisons across all our data sets have consistently shown five subconducting levels in addition to the fully open and closed levels for this channel.