Mechanosensitive Channel Piezo1 Research Articles

Greasing the Gears of Mechanosensitive Piezo Channels with Phosphoinositides and Cholesterol

Greasing the Gears of Mechanosensitive Piezo Channels with Phosphoinositides and Cholesterol

Structural mechanism for gating of a eukaryotic mechanosensitive channel of small conductance

Mechanosensitive ion channels transduce physical force into electrochemical signaling that underlies an array of fundamental physiological processes, including hearing, touch, proprioception, osmoregulation, and morphogenesis. The mechanosensitive channels of small conductance (MscS) constitute a remarkably diverse superfamily of channels critical for management of osmotic pressure. Here, we present cryo-electron microscopy structures of a MscS homolog from Arabidopsis thaliana, MSL1, presumably in both the closed and open states. The heptameric MSL1 channel contains an unusual bowl-shaped transmembrane region, which is reminiscent of the evolutionarily and architecturally unrelated mechanosensitive Piezo channels. Upon channel opening, the curved transmembrane domain of MSL1 flattens and expands. Our structures, in combination with functional analyses, delineate a structural mechanism by which mechanosensitive channels open under increased membrane tension. Further, the shared structural feature between unrelated channels suggests the possibility of a unified mechanical gating mechanism stemming from membrane deformation induced by a non-planar transmembrane domain.

Mechanosensitive Piezo Channels in Cancer: Focus on altered Calcium Signaling in Cancer Cells and in Tumor Progression.

Mechanotransduction, the translation of mechanical stimuli into biological signals, is a crucial mechanism involved in the function of fundamentally all cell types. In many solid tumors, the malignant transformation is often associated with drastic changes in cell mechanical features. Extracellular matrix stiffness, invasive growth, and cell mobility are just a few hallmarks present in cancer cells that, by inducing mechanical stimuli, create positive feedbacks promoting cancer development. Among the molecular players involved in these pathophysiological processes, the mechanosensitive Ca2+-permeable Piezo channels have emerged as major transducers of mechanical stress into Ca2+ dependent signals. Piezo channels are overexpressed in several cancers, such as in breast, gastric, and bladder, whereas their downregulation has been described in other cancers. Still, the roles of mechanosensitive Piezos in cancer are somewhat puzzling. In this review, we summarize the current knowledge on the pathophysiological roles of these Ca2+-permeable channels, with special emphasis on their functional involvement in different cancer types progression.

A Plug-and-Latch Mechanism for Gating the Mechanosensitive Piezo Channel

The mechanosensitive Piezo1 and Piezo2 channels convert mechanical force into cation permeation. However, their precise mechanogating and regulatory mechanisms remain elusive. Here, we report that Piezo1 utilizes three lateral ion-conducting portals equipped with physical gates for cooperative gating and splicing regulation. Mutating residues lining the portal converts Piezo1 into an anion-selective channel, demonstrating the portal-based cation-permeating pathway. Intriguingly, the portal is physically blocked with a plug domain, which undergoes alternative splicing in both Piezo1 and Piezo2. The Piezo1 isoform has local openings of the portals, enlarged single-channel conductance and sensitized mechanosensitivity. Remarkably, the three plugs are strategically latched onto the central axis for coordinated gating of the three portals. Disrupting the latching induces three quantal sub-conductance states in Piezo1, but not in the isoform. Together, we propose that Piezo utilizes an elegant plug-and-latch mechanism to physically and coordinately gate the lateral portals through the spliceable plug gates.

Structure and Mechanogating of the Mechanosensitive PIEZO Channel

The evolutionarily conserved PIEZO family of proteins, including PIEZO1 and PIEZO2, forms the long-sought bona fide mammalian mechanosensitive cation channel, and plays critical roles in various mechanotransduction processes such as touch, proprioception, tactile pain, vascular development and blood pressure regulation. Taking a multidisciplinary approach combining protein engineering and purification, cryo-EM, electrophysiology and drug screening, we have determined the three-bladed, propeller-like trimeric structures of both PIEZO1 and PIEZO2 that comprises 114 transmembrane helices (38 per protomer). Transmembrane helices 1-36 (TM1-36) are folded into nine tandem transmembrane helical units (THU) of four TMs each to form the unusual non-planar blades. The three blades are collectively curved into a nano-bowl shape of 28-nm diameter and 10-nm depth, with an extracellular cap-like structure embedded in the centre and a 9-nm-long intracellular beam connecting to the central pore. TM38 and the C-terminal domain are surrounded with the anchor domain and TM37, and enclose the central pore with both transmembrane and cytoplasmic constriction sites. Structural comparison between PIEZO1 and PIEZO2 has revealed that the transmembrane constriction site might act as a transmembrane gate. Structural and functional analyses have suggested that the PIEZO channel might employ the top cap to control the transmembrane gate, while the blade-beam might function as a lever-like apparatus to allosterically control the putative cytosolic gate, indicating a dual-gating mechanism. Together, our studies provide insights into the structure and mechanogating mechanism of PIEZO channels.

Characterizing the Expression and Function of the Mechanosensitive Piezo1 Channel in the Heart

Characterizing the Expression and Function of the Mechanosensitive Piezo1 Channel in the Heart

The Emerging Role of Mechanosensitive Piezo Channels in Migraine Pain.

Recently discovered mechanosensitive Piezo channels emerged as the main molecular detectors of mechanical forces. The functions of Piezo channels range from detection of touch and pain, to control of the plastic changes in different organs. Recent studies suggested the role of Piezo channels in migraine pain, which is supposed to originate from the trigeminovascular nociceptive system in meninges. Interestingly, migraine pain is associated with such phenomenon as mechanical hypersensitivity, suggesting enhanced mechanotransduction. In the current review, we present the data that propose the implication of Piezo channels in migraine pain, which has a distinctive pulsatile character. These data include: (i) distribution of Piezo channels in the key elements of the trigeminovascular nociceptive system; (ii) the prolonged functional activity of Piezo channels in meningeal afferents providing a mechanistical basis for mechanotransduction in nociceptive nerve terminals; (iii) potential activation of Piezo channels by shear stress and pulsating blood flow; and (iv) modulation of these channels by emerging chemical agonists and modulators, including pro-nociceptive compounds. Achievements in this quickly expanding field should open a new road for efficient control of Piezo-related diseases including migraine and chronic pain.

Calcium-Permeable Channels in Tumor Vascularization: Peculiar Sensors of Microenvironmental Chemical and Physical Cues.

Calcium (Ca2+)-permeable channels are key players in different processes leading to blood vessel formation via sprouting angiogenesis, including endothelial cell (EC) proliferation and migration, as well as in controlling vascular features which are typical of the tumor vasculature.In this review we present an up-to-date and critical view on the role of Ca2+-permeable channels in tumor vascularization, emphasizing on the dual communication between growth factors (mainly VEGF) and Ca2+ signals. Due to the complexity of the tumor microenvironment (TME) as a source of multiple stimuli acting on the endothelium, we aim to discuss the close interaction between chemical and physical challenges (hypoxia, oxidative stress, mechanical stress) and endothelial Ca2+-permeable channels, focusing on transient receptor potential (TRP), store-operated Ca2+ channels (SOCs), and mechanosensitive Piezo channels. This approach will depict their crucial contribution in regulating key properties of tumor blood vessels, such as recruitment of endothelial progenitors cells (EPCs) in the early steps of tumor vascularization, abnormal EC migration and proliferation, and increased vascular permeability. Graphical abstract depicting the functional role of Ca2+-permeable TRP, SOCs and Piezo channels in the biological processes regulating tumor angiogenesis in presence of both chemical (oxidative stress and oxygen levels) and mechanical stimuli (ECM stiffness). SOCs store-operated Ca2+ channels, TRPA transient receptor potential ankyrin, TRPV transient receptor potential vanilloid, TRPC transient receptor potential canonical, TRPM transient receptor potential melastatin, TRPM transient receptor potential vanilloid, O2 oxygen, ECM extracellular matrix.

A mechanism for the activation of the mechanosensitive Piezo1 channel by the small molecule Yoda1

Mechanosensitive Piezo1 and Piezo2 channels transduce various forms of mechanical forces into cellular signals that play vital roles in many important biological processes in vertebrate organisms. Besides mechanical forces, Piezo1 is selectively activated by micromolar concentrations of the small molecule Yoda1 through an unknown mechanism. Here, using a combination of all-atom molecular dynamics simulations, calcium imaging and electrophysiology, we identify an allosteric Yoda1 binding pocket located in the putative mechanosensory domain, approximately 40 Å away from the central pore. Our simulations further indicate that the presence of the agonist correlates with increased tension-induced motions of the Yoda1-bound subunit. Our results suggest a model wherein Yoda1 acts as a molecular wedge, facilitating force-induced conformational changes, effectively lowering the channel’s mechanical threshold for activation. The identification of an allosteric agonist binding site in Piezo1 channels will pave the way for the rational design of future Piezo modulators with clinical value.

Non-Contact High-Frequency Ultrasound Microbeam Stimulation: A Novel Finding and Potential Causes of Cell Responses.

Invasiveness research is an essential step in breast cancer metastasis. The application of high-frequency ultrasound microbeam stimulation (HFUMS) offers a manner of determining the invasion potential of human breast cancer cells by eliciting the elevation of transient cytoplasmic calcium ions (Ca2+). The fluorescent index (FI), which is a composite parameter reflecting calcium elevations elicited by HFUMS, was shown to be higher in invasive breast cancer cells (MDA-MB-231) compared to weakly invasive breast cancer cells (MCF-7) using the low-intensity 50-MHz HFUMS. This novel finding shows significant difference from the reported studies in which MCF-7 cells showed no response to HFUMS. In addition to the negligible response of normal human breast cells (MCF-10A), HFUMS shows the potential to be capable of differentiating the normal cells from the cancer cells. To understand the mechanism of HFUMS worked on mechanotransduction in cells, different channel blockers were used to investigate the roles of specific channels during HFUMS. It was found that GsMTx4 (30 μM), a selective blocker of mechanosensitive Piezo channels, reduces the FI values significantly in MDA-MB-231 cells, while SKF-96365 (40 μM), a general TRP channel blocker, cannot induce the significant inhibition of FI values. The results indicate that Piezo channels may play the main role in invasion and metastatic propagation of cells.

The mechanosensitive Piezo1 channel is required for bone formation.

Mechanical load of the skeleton system is essential for the development, growth, and maintenance of bone. However, the molecular mechanism by which mechanical stimuli are converted into osteogenesis and bone formation remains unclear. Here we report that Piezo1, a bona fide mechanotransducer that is critical for various biological processes, plays a critical role in bone formation. Knockout of Piezo1 in osteoblast lineage cells disrupts the osteogenesis of osteoblasts and severely impairs bone structure and strength. Bone loss that is induced by mechanical unloading is blunted in knockout mice. Intriguingly, simulated microgravity treatment reduced the function of osteoblasts by suppressing the expression of Piezo1. Furthermore, osteoporosis patients show reduced expression of Piezo1, which is closely correlated with osteoblast dysfunction. These data collectively suggest that Piezo1 functions as a key mechanotransducer for conferring mechanosensitivity to osteoblasts and determining mechanical-load-dependent bone formation, and represents a novel therapeutic target for treating osteoporosis or mechanical unloading-induced severe bone loss.

Shear stress-induced nuclear shrinkage through activation of Piezo1 channels in epithelial cells.

The cell nucleus responds to mechanical cues with changes in size, morphology and motility. Previous work has shown that external forces couple to nuclei through the cytoskeleton network, but we show here that changes in nuclear shape can be driven solely by calcium levels. Fluid shear stress applied to MDCK cells caused the nuclei to shrink through a Ca2+-dependent signaling pathway. Inhibiting mechanosensitive Piezo1 channels through treatment with GsMTx4 prevented nuclear shrinkage. Piezo1 knockdown also significantly reduced the nuclear shrinkage. Activation of Piezo1 with the agonist Yoda1 caused similar nucleus shrinkage in cells not exposed to shear stress. These results demonstrate that the Piezo1 channel is a key element for transmitting shear force input to nuclei. To ascertain the relative contribution of Ca2+ to cytoskeleton perturbation, we examined F-actin reorganization under shear stress and static conditions, and showed that reorganization of the cytoskeleton is not necessary for nuclear shrinkage. These results emphasize the role of the mechanosensitive channels as primary transducers in force transmission to the nucleus.

Piezo1 Channels Contribute to Flow‐Induced Intracellular Calcium Transients in Mouse Cortical Collecting Duct (CCD)

Within the CCD, an acute increase in tubular fluid flow rate (TFFR) (i) subjects principal (PC) and intercalated (IC) cells therein to biomechanical forces including hydrostatic pressure/circumferential stretch (CS), fluid shear stress (FSS) and drag/torque on PC cilia, and (ii) leads to a biphasic increase in intracellular Ca2+ concentration ([Ca2+]i) characterized by an immediate high amplitude increase to a peak value within ~10 s due to release of IP3‐sensitive internal Ca2+ stores coupled to extracellular Ca2+ entry at the basolateral membrane, followed by a decay to a plateau elevation in [Ca2+]i that exceeds baseline, sustained by luminal Ca2+ entry (Liu et al. AJP Renal Physiology 2003, 2005; 2007). The latter increase in [Ca2+]i is necessary for flow induced K+ secretion (FIKS) in the microperfused mammalian CCD. The identity of the basolateral Ca2+ entry path is unknown. We sought to examine whether Piezo channels, a family of mechanically‐activated Ca2+ permeable channels, contribute to this response. RNAscope detected single Piezo1 RNA molecules in mouse renal cortex. Immunofluorescence studies in mice expressing Piezo1‐tdTomato construct revealed robust basolateral and faint apical Piezo1 localization in both PC and IC. Flow‐induced [Ca2+]i transients, inferred from fluorescence intensity ratios (FIRs), were measured in microperfused mouse CCDs loaded with the Ca2+ indicator fura‐2 and then subject to low or high TFFRs, in the absence or presence of GsMTx4 (piezo‐blocker) or Yoda1 (piezo1‐activator). IC in CCDs from male and female mice preincubated with luminal or bath GsMTx4 (5 μM) failed to exhibit a flow‐induced increase in [Ca2+]i in response to an acute increase in TFFR. Addition of luminal or bath Yoda 1 (1 μM) to CCDs perfused at a low TFFR led to an increase in [Ca2+]i over 90 s in both PC and IC; addition of Yoda1 (10 μM) to the bath further increased [Ca2+]i. We speculate that mechanosensitive Piezo1 channels, expressed on the basolateral>apical membranes of PC and IC in the mouse CCD, contribute to the increase in [Ca2+]i triggered by an increase in TFFR.Support or Funding InformationNIDDK: R01 DK38470, R01 DK51391 and P30 DK079307This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelial Upregulation of Mechanosensitive Channel Piezo1 in Pulmonary Hypertension

RationaleThe Piezo channel family (Piezo1 and Piezo2), one of the most important family of mechanosensing ion channels. Surprisingly, there are no studies on Piezo in the pulmonary hypertension. We focus on Piezo1 because of its imprtance in vascular deveopment, especially in endothelial cells. Our working hypothesis is that upregulated Piezo1 may be a critical pathogenic mechanism that contributes to augmented Ca2+ influx and excessive PAEC proliferation or apoptosis in patients with IPAH.MethodsRNA levels of Piezo1 and Piezo2 was quantified by RT‐PCR Analysis. Protein levels of Piezo1, bax, bcl‐2, Calpain‐1 and Calpain‐2 was quantified by Western blot analysis. adult Male C57BL/6 mice (6–8 weeks) were exposed to persistent chronic hypoxia (10% O2) for 21 days to establish hypoxia‐induced PH. For Sugen 5416/hypoxia (SuHx) induced‐PH rat model, SD rats (age, 6–8 weeks old) were either injected s.c. with 20 mg·kg−1 SU5416 (Sigma‐Aldrich, St. Louis, MO, USA) or vehicle, exposed to hypoxic conditions for 3 weeks (10% O2) and returned to normoxic condition (21% O2) for 2 weeks. In the MCT group, a single intraperitoneal injection of MCT (60 mg/kg, Sigma Aldrich) was given at day 1 to establish the MCT‐induced PH.ResultsPiezo1 and Piezo2 have high expression in rat lung tissue and human PAEC, and also Piezo1 has significantly higher mRNA levels in both human PASMC and PAEC than Piezo2 (n=5, p<0.01). Upregulated Piezo1 in whole‐lung tissue (mainly containing ECs) from MCT‐mediated PH (MCT‐PH) compared to normal control rats (1.00±0.07; 4.74±0.41, n>5, p<0.001). Compared to controls, Piezo1 was increased in whole‐lung tissues in hypoxic mice (1.00±0.07; 4.74±0.41, n>5, p<0.01) and SuHx rats (1.00±0.15; 1.99±0.31, n>5, p<0.01). Also, compared to normoxia condition, increased Piezo1 was associated with a increased Calpain‐1 and Calpain‐2 treated with hypoxia (3% O2 for 72 hours) (1.00±0.16; 4.14±0.75; 1.00±0.14, 2.32±0.35; 1.00±0.07; 1.74±0.14, n>5, p<0.01). Moreover, PAECs from IPAH patients exhibited significant elevation of Piezo1 compared to normal control subjects (1.00±0.09; 6.20±1.78, n=5, p<0.01).ConclusionsThese results are the first to implicate that Piezo1 as a mechanosensor may play an important role in the development of pulmonary hypertension via, at least in part, induction of PAEC apoptosis and enhancement of Ca2+ in PAECs. Piezo1 may be involved in the transcriptional regulation under hypoxia and play critical roles in the development of PH. Therefore, further studies are needed to identify the potential contribution of Piezo1 in PH.Support or Funding InformationNone.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional expression of the mechanosensitive PIEZO1 channel in primary endometrial epithelial cells and endometrial organoids

Successful pregnancy requires the establishment of a complex dialogue between the implanting embryo and the endometrium. Knowledge regarding molecular candidates involved in this early communication process is inadequate due to limited access to primary human endometrial epithelial cells (EEC). Since pseudo-pregnancy in rodents can be induced by mechanical scratching of an appropriately primed uterus, this study aimed to investigate the expression of mechanosensitive ion channels in EEC. Poking of EEC provoked a robust calcium influx and induced an increase in current densities, which could be blocked by an inhibitor of mechanosensitive ion channels. Interestingly, RNA expression studies showed high expression of PIEZO1 in EEC of mouse and human. Additional analysis provided further evidence for the functional expression of PIEZO1 since stimulation with Yoda1, a chemical agonist of PIEZO1, induced increases in intracellular calcium concentrations and current densities in EEC. Moreover, the ion channel profile of human endometrial organoids (EMO) was validated as a representative model for endometrial epithelial cells. Mechanical and chemical stimulation of EMO induced strong calcium responses supporting the hypothesis of mechanosensitive ion channel expression in endometrial epithelial cells. In conclusion, EEC and EMO functionally express the mechanosensitive PIEZO1 channel that could act as a potential target for the development of novel treatments to further improve successful implantation processes.

Structural Bases for Chemical and Mechanical Gating in the Piezo1 Channel

Mechanosensitive Piezo channels possess a propeller-like structure with a central cation-selective pore and three putative force-sensing blades encompassing several helical bundles called Piezo repeats. How membrane stretch and chemical agonists modulate Piezo channels is unknown. Here, using all-atom molecular dynamic stimulations and experimental assays, we localize the binding site of Yoda1, a small molecule Piezo1 activator, and identify stretch-induced conformational rearrangements of the blades. Yoda1 binding uncouples two adjacent Piezo repeats, facilitating stretch-induced blade motions, and alters long-range residue-residue contacts between pore and blades, as evidenced by Yoda1-induced channel activation of a mechanically-insensitive mutant. In addition, cation-selective fenestrations allow potassium, not chloride, ions to enter a pore vestibule. Our work reveals the structural bases of cationic selectivity, chemical modulation and mechanical sensing in a mammalian Piezo channel.

Piezo1 Mediated Ca2+ Signaling Causes Nuclear Shrinkage under Fluid Shear Stress

Nuclei of cells respond to local mechanical cues with changes in their size, morphology, and motility, to maintain tissue homeostasis and proper functions. Previous studies indicated that the external forces are coupled to nuclei through the surrounding cytoskeleton network linked to the nuclear envelope, but the molecules that directly interact with outside environments are unclear. Here we show that the fluid shear stress applied to MDCK cells caused the nucleus to shrink through a Ca2+ dependent signaling pathway. Inhibiting Ca2+ permeable mechanosensitive Piezo1 channels, with the specific inhibitor GsMTx4, eliminated the nuclear changes. Piezo1 knockdown with miRNA that targets Piezo1 significantly reduced the nuclear shrinkage under shear stress. Activation of Piezo1 with Yoda1 caused similar nucleus shrinkage without shear stress. These results demonstrate that Piezo1 channel is accountable for transmitting the shear force input to nuclei via Ca2+ signaling. To assess the relative contributions of Ca2+ and cytoskeleton reorganization on force transduction, we examined F-actin reorganization under shear stress and static conditions, and showed that reorganization is not necessary for the nucleus shrinkage. These results emphasize the role of the mechanosensitive channels as primary transducers in force transmission to the nucleus.

Recent advances in our understanding of the structure and function of more unusual cation channels.

As their name implies, cation channels allow the regulated flow of cations such as sodium, potassium, calcium, and magnesium across cellular and intracellular membranes. Cation channels have long been known for their fundamental roles in controlling membrane potential and excitability in neurons and muscle. In this review, we provide an update on the recent advances in our understanding of the structure–function relationship and the physiological and pathophysiological role of cation channels. The most exciting developments in the last two years, in our opinion, have been the insights that cryoelectron microscopy has provided into the inner life and the gating of not only voltage-gated channels but also mechanosensitive and calcium- or sodium-activated channels. The mechanosensitive Piezo channels especially have delighted the field not only with a fascinating new type of structure but with important roles in blood pressure regulation and lung function.

A hydrophobic gate in the inner pore helix is the major determinant of inactivation in mechanosensitive Piezo channels.

Piezo1 and Piezo2 belong to a family of mechanically-activated ion channels implicated in a wide range of physiological processes. Mechanical stimulation triggers Piezo channels to open, but their characteristic fast inactivation process results in rapid closure. Several disease-causing mutations in Piezo1 alter the rate of inactivation, highlighting the importance of inactivation to the normal function of this channel. However, despite the structural identification of two physical constrictions within the closed pore, the mechanism of inactivation remains unknown. Here we identify a functionally conserved inactivation gate in the pore-lining inner helix of mouse Piezo1 and Piezo2 that is distinct from the two constrictions. We show that this gate controls the majority of Piezo1 inactivation via a hydrophobic mechanism and that one of the physical constrictions acts as a secondary gate. Our results suggest that, unlike other rapidly inactivating ion channels, a hydrophobic barrier gives rise to fast inactivation in Piezo channels.

The mechanosensitive Piezo1 channel: a three-bladed propeller-like structure and a lever-like mechanogating mechanism.

The evolutionarily conserved Piezo proteins, including Piezo1 and Piezo2, constitute a bona fide class of mechanosensitive (MS) cation channels, which play critical roles in various mammalian physiologies, including sensation of touch, proprioception and regulation of vascular development, and blood pressure. Furthermore, mutations in Piezos have been linked to various human genetic diseases, validating their potential as therapeutic targets. Thus, it is pivotal to understand how Piezo channels effectively convert mechanical force into selective cation permeation, and therefore precisely control the various mechanotransduction processes. On the basis of our recently determined cryoelectron microscopy structures of the full-length 2547-residue mouse Piezo1, structure-guided mutagenesis, and electrophysiological and pharmacological characterizations, here we focus on reviewing the key structural features and functional components that enable Piezo1 to employ a lever-like mechanogating mechanism to function as a sophisticated mechanotransduction channel.