Mechanosensitive Ion Channel Piezo1 Research Articles

Cholesterol-Dependent Piezo1 Clusters are Essential for Efficient Cellular Mechanotransduction

The human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis [1,2,3]. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichment, depletion (methyl-β-Cyclodextrin; MBCD) and the disruption of membrane cholesterol organization (Dynasore) affects PIEZO1's response to mechanical force. STORM super-resolution imaging revealed that, at the nano-scale, PIEZO1 channels in the membrane associate as clusters which increase in number upon cholesterol addition. Both cluster size and diffusion rates were profoundly affected by treatment with MBCD (5 mM). In addition, electrophysiological recordings in the cell-attached configuration revealed that MBCD caused a rightward shift in the PIEZO1 pressure-response curve and increased channel latency in response to mechanical stimuli. Our results indicate that PIEZO1 function is directly dependent on the amount and lateral organization of membrane cholesterol which is crucial for the concerted inactivation of PIEZO1 clusters. 1 Cahalan SM, et al. Piezo1 links mechanical forces to red blood cell volume. eLife. 2015;4. 2 Rode B, et al. Piezo1 channels sense whole body physical activity to reset cardiovascular homeostasis and enhance performance. Nat Commun. 2017;8. 3 Li J, et al. Piezo1 integration of vascular architecture with physiological force. Nature, 2014. Supported by the Australian Research Council and the National Health and Medical Research Council of Australia.

The Mechanosensitive Ion Channel Piezo1 Significantly Mediates Ultrasonic Stimulation of Neurons

Ultrasound brain stimulation is a promising modality for probing brain function and treating brain disease non-invasively and with high spatiotemporal resolution. However, the mechanism underlying its effects remains unclear. Here, we examine the role that the mouse piezo-type mechanosensitive ion channel component 1 (Piezo1) plays in mediating the effects of ultrasound in mouse primary cortical neurons and a neuronal cell line. We show that ultrasound alone could activate heterologous and endogenous Piezo1, initiating calcium influx and increased nuclear c-Fos expression in primary neurons, but not when pre-treated with a Piezo1 inhibitor. We also found that ultrasound significantly increased the expression of the important proteins phospho-CaMKII, phospho-CREB and c-Fos in a neuronal cell line, but knocking down Piezo1 significantly reduced this effect. Our findings demonstrate that the activity of mechanosensitive ion channels like Piezo1 stimulated by ultrasound is an important contributor to its neuro-stimulation effects.

Unlocking the Dangers of a Stiffening Brain

Mechanical cues regulate neuronal function and reactivity of glial cells, the origin of gliomas. In this issue of Neuron, Chen etal. (2018) uncover a feedforward loop mediated by the mechanosensitive ion channel Piezo1 and tissue stiffness that drives glioma aggression.

A Feedforward Mechanism Mediated by Mechanosensitive Ion Channel PIEZO1 and Tissue Mechanics Promotes Glioma Aggression

Alteration of tissue mechanical properties is a physical hallmark of solid tumors including gliomas. How tumor cells sense and regulate tissue mechanics is largely unknown. Here, we show that mechanosensitive ion channel Piezo regulates mitosis and tissue stiffness of Drosophila gliomas, but not non-transformed brains. PIEZO1 is overexpressed in aggressive humangliomas and its expression inversely correlates with patient survival. Deleting PIEZO1 suppresses the growth of glioblastoma stem cells, inhibits tumor development, and prolongs mouse survival. Focal mechanical force activates prominent PIEZO1-dependent currents from glioma cell processes, but not soma. PIEZO1 localizes at focal adhesions to activate integrin-FAK signaling, regulate extracellular matrix, and reinforce tissue stiffening. In turn, a stiffer mechanical microenvironment elevates PIEZO1 expression to promote glioma aggression. Therefore, glioma cells are mechanosensory in a PIEZO1-dependent manner, and targeting PIEZO1 represents a strategy to break the reciprocal, disease-aggravating feedforward circuit between tumor cell mechanotransduction and the aberrant tissue mechanics. VIDEO ABSTRACT.

Expression characteristics of Piezo1 protein in stress models of human degenerative chondrocytes

To explore the expression characteristics of new mechanosensitive ion channel Piezo1 protein in stress models of human degenerative chondrocytes. The stress stimulation model of human degenerative chondrocytes in vitro was constructed. Multi-channel cell stretch stress loading system FX-4000T was used to treat chondrocytes. According to the results of pre-test, the loading frequency of 0.5 Hz and the cell elongation of 20% were loaded. According to cell processing time, it was divided into 0 h, 2 h, 12 h, 24 h and 48 h mechanical stress group. The RT-PCR and Western-blot were used to test the expression of the Piezo1, also the Laser scanning confocal microscope (LSCM) was used to test the intensity of the fluorescence of the Piezo1. (1)The result of the RT-PCR showed that the expression of the Piezo1 in the 2 h group was higher than the 0 h group(F=13.917, q=0.037 1, P<0.05). The expression of the piezo1 in the 24 h group was the highest. While the expression of the piezo1 in the 48 h group was lower than the expression of the piezo1 in the 24 h group(F=13.917, q=0.049 5, P<0.05). (2)The result of the Western-blot showed that the 2 h group was higher than the 0 h group(F=19.341, q=0.037 1, P<0.05). The expression of the 24 h had the highest expression which was higher than the 48 h group(F=19.341, q=0.017 7, P<0.05). (3)The Piezo1 protein was extensively expressed in the cytoplasm and nucleus of the nucleus pulposus cells. And with the increase of stress processing time, the fluorescence intensity of the protein also increased. In human degeneration cartilage cells, the new mechanio sensitive ion channel Piezo1 protein has a trace expression. After loading periodic mechanical tensile force, the expression of Piezo1 protein increases with time dependence.

Sickle cell dehydration: Pathophysiology and therapeutic applications.

Cell dehydration is a distinguishing characteristic of sickle cell disease and an important contributor to disease pathophysiology. Due to the unique dependence of Hb S polymerization on cellular Hb S concentration, cell dehydration promotes polymerization and sickling. In double heterozygosis for Hb S and C (SC disease) dehydration is the determining factor in disease pathophysiology. Three major ion transport pathways are involved in sickle cell dehydration: the K-Cl cotransport (KCC), the Gardos channel (KCNN4) and Psickle, the polymerization induced membrane permeability, most likely mediated by the mechano-sensitive ion channel PIEZO1. Each of these pathways exhibit unique characteristics in regulation by oxygen tension, intracellular and extracellular environment, and functional expression in reticulocytes and mature red cells. The unique dependence of K-Cl cotransport on intracellular Mg and the abnormal reduction of erythrocyte Mg content in SS and SC cells had led to clinical studies assessing the effect of oral Mg supplementation. Inhibition of Gardos channel by clotrimazole and senicapoc has led to Phase 1,2,3 trials in patients with sickle cell disease. While none of these studies has resulted in the approval of a novel therapy for SS disease, they have highlighted the key role played by these pathways in disease pathophysiology.

Mechanosensitive ion channel Piezo1 is expressed in antral G cells of murine stomach

G cells in the antrum region of the murine stomach produce gastrin, the central hormone for controlling gastric activities. Secretion of gastrin is induced mainly by protein breakdown products but also by distensions of the stomach wall. Although G cells respond to protein fragments via distinct chemosensory receptor types, the mechanism underlying G cell activation upon distention is entirely ambiguous. Mechanosensitive ion channels are considered as potential candidates for such a task. Therefore, we explore the possibility of whether Piezo1, a polymodal sensor for diverse mechanical forces, is expressed in antral G cells. The experimental analyses revealed that the vast majority of G cells indeed expressed Piezo1. Within flask-like G cells at the base of the antral invaginations, the Piezo1 protein was primarily located at the basolateral portion, which is thought to be the release site for the exocytic secretion of gastrin. In the spindle-like G cells, which are oriented parallel to the invaginations, Piezo1 protein was restricted to the cell body where the hormone was also located, whereas the long processes appeared to be devoid of Piezo1 protein. Our results suggest that mechanosensitive channels such as Piezo1, located in close proximity to hormone-release sites, enable G cells to respond directly to antrum distensions with gastrin secretion.

Chondroprotection by urocortin involves blockade of the mechanosensitive ion channel Piezo1

Osteoarthritis (OA) is characterised by progressive destruction of articular cartilage and chondrocyte cell death. Here, we show the expression of the endogenous peptide urocortin1 (Ucn1) and two receptor subtypes, CRF-R1 and CRF-R2, in primary human articular chondrocytes (AC) and demonstrate its role as an autocrine/paracrine pro-survival factor. This effect could only be removed using the CRF-R1 selective antagonist CP-154526, suggesting Ucn1 acts through CRF-R1 when promoting chondrocyte survival. This cell death was characterised by an increase in p53 expression, and cleavage of caspase 9 and 3. Antagonism of CRF-R1 with CP-154526 caused an accumulation of intracellular calcium (Ca2+) over time and cell death. These effects could be prevented with the non-selective cation channel blocker Gadolinium (Gd3+). Therefore, opening of a non-selective cation channel causes cell death and Ucn1 maintains this channel in a closed conformation. This channel was identified to be the mechanosensitive channel Piezo1. We go on to determine that this channel inhibition by Ucn1 is mediated initially by an increase in cyclic adenosine monophosphate (cAMP) and a subsequent inactivation of phospholipase A2 (PLA2), whose metabolites are known to modulate ion channels. Knowledge of these novel pathways may present opportunities for interventions that could abrogate the progression of OA.

Urocortin protects chondrocytes from acute trauma in articular cartilage

Purpose: Single injurious mechanical impact to joints, as seen in sports injury, is known to be associated with a high risk of later development of osteoarthritis (OA). Irreversible chondrocyte death is initiated following impact and results in progressive breakdown of articular cartilage. Promotion of chondrocyte survival is therefore a necessary target in the prevention of development of further OA. Urocortin1 (Ucn) has recently been shown to have a pro-survival effect on chondrocytes in both the presence and absence of pro-apoptotic stimuli. The aim of this study was to investigate the role of Ucn in response to mechanical impact. Methods: Full thickness porcine cartilage explants from lateral and medial femoral condyles were impacted with a 500g weight using a custom-made drop tower device. Impacted explants were treated with human Ucn (10-7M, 30 minutes prior to or following impact), non-selective cation channel blocker Gadolinium (500μM, 30 minutes following impact), or mechanosensitive ion channel blocker GsMTx4 (40μM, 2 hours prior to impact) and cultured for 72hrs. Cell viability was assessed with a CMFDA:PI live dead assay, using both macro confocal microscopy, as a means of avoiding bias, and high magnification confocal microscopy techniques. Lactate dehydrogenase (LDH) assays were conducted at 72hrs to provide a quantitative read out of cell death for each explant. Calcium influx was assessed 1hr post impact by Fluo-4 AM staining and visualised by multiphoton microscopy. Results: Porcine explants were impacted from a range of heights (10-70mm) in order to establish the correct energy of impact for inducing cell death without excess damage and fracture of the cartilage explant. Cell death increased in a dose dependent manner with height. 50mm was chosen as optimal height for all further experiments. Pre-treatment of explants with Ucn had a significant increase on cell viability when compared to impacted alone, as seen by quantification of the CMFDA:PI ratio on macro confocal (p=0.0042) and by high magnification confocal microscopy (p=0.0015). This increase in cell viability was confirmed by LDH assay (p=0.0063). Post-impact treatment of the explants with Ucn also resulted in a significant increase in cell survival, seen in both CMFDA:PI ratio (p=0.0009) and LDH assay (p=0.0053). We have previously found in primary human chondrocytes that Ucn exerts its protective effect on chondrocytes by the inhibition of excessive calcium influx. Calcium influx was therefore visualised in impacted explants treated pre- and post-impact with Ucn. A significant decrease in the levels of calcium staining was seen in both of these conditions when compared to explants impacted in the absence of Ucn (p=0.0073, p=0.05). In vitro work also highlighted a role for the mechanosensitive ion channel Piezo1 in cell death associated with the absence of Ucn. Explants were therefore treated with both Gadolinium and GsMTx4 post-impact and cell viability assessed by multiphoton microscopy. An increase in cell viability was observed in both conditions, comparable to those seen with addition of Ucn. Conclusions: A novel role for Ucn in the protection of articular chondrocytes maintained in cartilage explants has been shown and validated by two microscopy techniques and LDH assay. Ucn has also been shown to reduce calcium influx in impacted explants at 60 minutes post impact, and increased cell viability was observed by blocking mechanosensitive ion channels. These data suggest the importance of regulation of calcium influx in Ucn-mediated cell protection. The pro-survival effect of Ucn was seen both pre- and post-impact, highlighting the potential prophylactic and therapeutic use of Ucn for treating sudden injury to cartilage, and subsequent progression to post-traumatic OA.

Human Piezo1 Membrane Localization and Gating Kinetics are Modulated by Cholesetrol Levels

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.

Mechanosensing is critical for axon growth in the developing brain.

During nervous system development, neurons extend axons along well-defined pathways. The current understanding of axon pathfinding is based mainly on chemical signalling. 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 (RGC) axons. In vivo atomic force microscopy revealed striking stiffness gradient patterns in the embryonic brain. RGC axons grew towards the tissue’s softer side, 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.

Functional role of mechanosensitive ion channel Piezo1 in human periodontal ligament cells.

To evaluate the function of Piezo1, an evolutionarily conserved mechanically activated channel, in periodontal ligament (PDL) tissue homeostasis under compressive loading. Primary human PDL cells (hPDLCs) were isolated, cultured, and then subjected to 2.0 g/cm(2) static compressive loading for 0.5, 3, 6, and 12 hours, respectively. The expressions of Piezo1 and osteoclastogenesis marker gene were assessed by semiquantitative reverse transcription-polymerase chain reaction. In addition, Piezo1 inhibitor, GsMTx4, was used to block the function of Piezo1, and tumor necrosis factor-α was also used as a positive control. After 12 hours of compressive loading the PDLCs were co-cultured with murine monocytic cell line RAW264.7. Immunofluorescence, western blot, enzyme-linked immunosorbent assay, and tartrate-resistant acid phosphatase staining were also used to test the potency of PDLCs to induce osteoclastogenesis and the activation of nuclear factor (NF)-κB. Piezo1, cyclooxygenase-2, receptor activator of NF-κB ligand, and prostaglandin E2 were significantly upregulated under static compressive stimuli. GsMTx4 repressed osteoclastogenesis in the mechanical stress-pretreated PDLCs-RAW264.7 co-culture system. Furthermore, NF-κB signaling pathway was involved in the mechanical stress-induced osteoclastogenesis. Piezo1 exerts a transduction role in mechanical stress-induced osteoclastogenesis in hPDLCs.

Positively Charged Residues on GsMTx4 are Crucial for Inhibition of the Mechanosensitive Ion Channel Piezo1

GsMTx4 is the only known peptide inhibitor of mechanosensitive channels. It is a gating modifier and presumably acts by modifying the arrangement of lipid molecules in the channel-lipid interface. Like other ICK peptide toxins GsMTx4 has a hydrophobic face which presumably facilitates partitioning of the molecule to the water/membrane interface of the bilayer. Prior studies also show that charged lysine residues may further influence the positioning of the peptide in the bilayer through interactions with the headgroups of anionic lipids. Coarse-grained simulations of GsMTx4 in the lipid bilayer suggest the existence of two binding modes: a shallow-binding mode and a deep-binding mode. The deep-binding mode in particular involves interactions between positively charged residues on the peptide and carbonyl atoms of the headgroups of inner-leaflet lipids. To provide insight into these interactions for GsMTx4 function, we investigated if reversing the charge by mutating lysine residues to glutamate affected the activity of GsMTx4. Each lysine residue in GsMTx4 was individually changed to glutamate to generate six mutants: K8E, K15E, K20E, K22E, K25E and K28E. When tested on outside-out patches from HEK cells transfected with mPiezo1 cDNA, all KtoE mutants showed reduced ability to inhibit the channel. In addition to reduced activity, K15E peptide altered the inactivation rate of the mPiezo1 currents. Even at saturating concentrations, K28E mutation reduced the activity of the peptide to 23% of that of the wild-type peptide. This study shows that charge reversing mutations to lysine residues diminish the efficacy of GsMTx4, and this phenomenon may involve disruption of peptide partitioning into the membrane and/or peptide-induced bending of the membrane bilayer.

The Mechanosensitive Ion Channel Piezo1 Is Inhibited by the Peptide GsMTx4

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.