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PIEZO1 encodes a mechanoreceptor, a cation channel activated by mechanical stimuli. Gain-of-function (GoF) variants in PIEZO1 cause dehydrated hereditary stomatocytosis (DHS), or xerocytosis, a pleiotropic syndrome characterized by anemia and iron overload. DHS patients develop hepatic iron overload independent of the degree of anemia and transfusion regimen. PIEZO1-GoF variants suppress hepcidin expression in both hepatic cellular model and constitutive/macrophage-specific Piezo1-GoF mice model. Therefore, PIEZO1-GoF variants regulate hepcidin expression by a crosstalk between hepatocytes (HCs) and macrophages with a still unknown mechanism. Transcriptomic and proteomics analysis in the human hepatic Hep3B cells engineered for the PIEZO1-R2456H variant (PIEZO1-KI) revealed alterations in the actin cytoskeleton regulation, MAPK cascade, and RAS signaling. These changes mainly occur through a novel key regulator, RRAS, whose protein and mRNA levels are regulated by PIEZO1 activation and inhibition. This regulation was further confirmed in C57BL/6 mouse primary HCs treated with Yoda-1 and/or GsMTx-4. Indeed, PIEZO1-KI cells exhibited hyper-activated RAS-GTPase activity that is rescued by PIEZO1 inhibition, restoring expression of the hepcidin gene HAMP. A negative correlation between RAS signaling and HAMP regulation was confirmed by inhibiting RAS-GTPase and MEK1-2 activity. Conversely, rescued HAMP gene expression requires downregulation of RRAS, confirming negative feedback between RAS-MAPK and BMP/SMADs pathways in HAMP regulation. We demonstrated that PIEZO1-GoF variants influence the actin cytoskeleton organization by activating the hepatic RAS signaling system. Understanding the role of RAS signaling in regulating iron metabolism could pave the way for new therapeutic strategies in DHS and other conditions characterized by iron overload., (© 2024 The Author(s). American Journal of Hematology published by Wiley Periodicals LLC.)

From haematopoietic stem cells to megakaryocytes (Mks), cells undergo various mechanical forces that affect Mk differentiation, maturation and proplatelet formation. The mechanotransductor PIEZO1 appears to be a natural candidate for sensing these mechanical forces and regulating megakaryopoiesis and thrombopoiesis. Gain-of-function mutations of PIEZO1 cause hereditary xerocytosis, a haemolytic anaemia associated with thrombotic events. If some functions of PIEZO1 have been reported in platelets, few data exist on PIEZO1 role in megakaryopoiesis. To address this subject, we used an in vitro model of Mk differentiation from CD34 + cells and studied step-by-step the effects of PIEZO1 activation by the chemical activator YODA1 during Mk differentiation and maturation. We report that PIEZO1 activation by 4 μM YODA1 at early stages of culture induced cytosolic calcium ion influx and reduced cell maturation. Indeed, CD41 + CD42 + numbers were reduced by around 1.5-fold, with no effects on proliferation. At later stages of Mk differentiation, PIEZO1 activation promoted endomitosis and proplatelet formation that was reversed by PIEZO1 gene invalidation with a shRNA-PIEZO1. Same observations on endomitosis were reproduced in HEL cells induced into Mks by PMA and treated with YODA1. We provide for the first time results suggesting a dual role of PIEZO1 mechanotransductor during megakaryopoiesis., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Hereditary erythrocytes disorders include a large group of conditions with heterogeneous molecular bases and phenotypes. We analyzed here a case series of 155 consecutive patients with clinical suspicion of hereditary erythrocyte defects referred to the Medical Genetics Unit from 2018 to 2020. All of the cases followed a diagnostic workflow based on a targeted next-generation sequencing panel of 86 genes causative of hereditary red blood cell defects. We obtained an overall diagnostic yield of 84% of the tested patients. Monogenic inheritance was seen for 69% (107/155), and multi-locus inheritance for 15% (23/155). PIEZO1 and SPTA1 were the most mutated loci. Accordingly, 16/23 patients with multi-locus inheritance showed dual molecular diagnosis of dehydrated hereditary stomatocytosis/xerocytosis and hereditary spherocytosis. These dual inheritance cases were fully characterized and were clinically indistinguishable from patients with hereditary spherocytosis. Additionally, their ektacytometry curves highlighted alterations of dual inheritance patients compared to both dehydrated hereditary stomatocytosis and hereditary spherocytosis. Our findings expand the genotypic spectrum of red blood cell disorders and indicate that multi-locus inheritance should be considered for analysis and counseling of these patients. Of note, the genetic testing was crucial for diagnosis of patients with a complex mode of inheritance.

Background: Dehydrated hereditary stomatocytosis (DHS) or hereditary xerocytosis is a rare, autosomal dominant hemolytic anemia characterized by macrocytosis, presence of stomatocytes and dehydration of red blood cells (RBCs). The dehydration is caused by a defect in cellular cation content. The most frequent expression of the pathology is hemolytic well-compensated anemia with high reticulocyte count, a tendency to macrocytosis, increased mean corpuscular hemoglobin concentration (MCHC) and mild jaundice. We here describe a new mutation of PIEZO1 gene, the most frequent mutated gene in DHS, in a family affected by hereditary hemolytic anemia., Case Presentation: We describe the case of a 12-years-old girl with well-compensated chronic hemolysis, increased MCHC and a father who had the same hematological characteristics. After excluding secondary causes of chronic hemolysis and enzymatic defects of the RBCs, microscopic observation of the peripheral blood smear, tests of RBC lysis, ektacytometry, SDS-PAGE and in last instance genetic analysis has been performed. This complex diagnostic workup identified a new variant in the PIEZO1 gene, never described in literature, causative of DHS. This pathogenetic variant was also detected in the father., Conclusions: This case report highlights the importance of a correct and exhaustive diagnostic-workup in patients with clinical suspicious for hemolytic anemia in order to make a differential diagnosis. This is relevant for the management of these patients because splenectomy is contraindicated in DHS due to high thrombotic risk.

Hereditary Xerocytosis (HX) is an autosomal dominantly inherited congenital hemolytic anemia associated with erythrocyte dehydration due to decreased intracellular potassium content resulting in increased mean corpuscular hemoglobin concentration. The affected members of HX families show compensated anemia with splenomegaly, hemosiderosis, and perinatal edema but are in large part transfusion independent. Functional studies show a link between mutations in mechanosensitive ion channel, encoded by PIEZO1 gene and the HX. We identified new PIEZO1 variants that are likely pathogenic in three phenotypically characterized multi-generational HX Brazilian families. Interestingly, one missense variant of the PIEZO1 gene identified, p.E2494V was associated in trans with the previously reported most frequent pathogenic duplication p.E2496ELE. The three-dimensional structure of the human protein modeled using structural coordinates of the mouse Piezo1 solved by cryo-electron microscopy (Cryo-ME) showed that the two identified variants, p.M2007L and p.T2014I, are localized to an important mechanosensitive transmembrane domain suggesting a conformational mechanism for altered channel's gating. The p.E2496ELE variant identified alters the extension of helix α1 bringing it much closer to the beam affecting the position of it structure at the end of the pore., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Hereditary xerocytosis (HX), also known as dehydrated stomatocytosis (DHSt) is a dominantly inherited genetic disorder exhibiting red cell membrane dehydration caused by the loss of the monovalent cation K + and water. Variants in mechanosensitive Piezo ionic channels of the PIEZO1 gene are the primary cause of HX. We have utilized high throughput and highly precise next-generation sequencing (NGS) to make a diagnosis and examine the genotype-phenotype relationship in inflexible HX cases. Seven unrelated patients with unexplained hemolytic anemia were scrutinized with a panel probing 8000 genes related to congenital anemia. Targeted next-generation sequencing identified 8 missense variants in the PIEZO1 gene in 7 unrelated Indian patients. Three of the 8 variants are novel (c.1795G > C, c.2915G > A, c.7372 T > C) and the remaining five (c.4082A > G, c.6829C > A, c.7374C > G, c.7381G > A, c.7483_7488dup) are previously reported. The variants have been validated by Sanger sequencing. One patient with autosomal dominant mutation (c.7372 T > C) is associated with iron refractory iron deficiency anemia. Of the 7 patients, one has HX in combination with a novel homozygous variant (c.994G > A) in the PKLR gene causing PK deficiency resulting in severe clinical manifestations with phenotypic variability. In silico prediction using bioinformatics tools were used to study the possible damaging effects of the novel variants. Structural-functional analysis of the novel variants was investigated by molecular modeling software (PyMOL and Swiss PDB). These results encompass the heterogeneous behavior of mechano-sensitive Piezo1 protein observed in HX patients in India. Moreover, NGS imparted a subtle, economical, and quick tool for understanding the genetic cause of undiagnosed cases of congenital hemolytic anemia. NGS grants a potential technology integrating clinical history together with molecular report profiting in such patients and their families.

Hereditary xerocytosis is a dominantly inherited red cell membrane disorder caused in most cases by gain-of-function mutations in PIEZO1, encoding a mechanosensitive ion channel that translates a mechanic stimulus into calcium influx. We found that PIEZO1 was expressed early in erythroid progenitor cells, and investigated whether it could be involved in erythropoiesis, besides having a role in the homeostasis of mature red cell hydration. In UT7 cells, chemical PIEZO1 activation using YODA1 repressed glycophorin A expression by 75%. This effect was PIEZO1-dependent since it was reverted using specific short hairpin-RNA knockdown. The effect of PIEZO1 activation was confirmed in human primary progenitor cells, maintaining cells at an immature stage for longer and modifying the transcriptional balance in favor of genes associated with early erythropoiesis, as shown by a high GATA2/GATA1 ratio and decreased α/β-globin expression. The cell proliferation rate was also reduced, with accumulation of cells in G0/G1 of the cell cycle. The PIEZO1-mediated effect on UT7 cells required calcium-dependent activation of the NFAT and ERK1/2 pathways. In primary erythroid cells, PIEZO1 activation synergized with erythropoietin to activate STAT5 and ERK, indicating that it may modulate signaling pathways downstream of erythropoietin receptor activation. Finally, we studied the in-vitro erythroid differentiation of primary cells obtained from 14 PIEZO1 -mutated patients, from 11 families, carrying ten different mutations. We observed a delay in erythroid differentiation in all cases, ranging from mild (n=3) to marked (n=8). Overall, these data demonstrate a role for PIEZO1 during erythropoiesis, since activation of PIEZO1 - both chemically and through activating mutations - delays erythroid maturation, providing new insights into the pathophysiology of hereditary xerocytosis., (Copyright© 2020 Ferrata Storti Foundation.)

Dehydrated hereditary stomatocytosis (DHS), or xerocytosis, is an autosomal dominant hemolytic anemia. Most patients with DHS carry mutations in the PIEZO1 gene encoding a mechanosensitive cation channel. We here demonstrate that patients with DHS have low levels of hepcidin and only a slight increase of ERFE, the erythroid negative regulator of hepcidin. We demonstrated that at the physiological level, PIEZO1 activation induced Ca 2+ influx and suppression of HAMP expression in primary hepatocytes. In two hepatic cellular models expressing PIEZO1 WT and two PIEZO1 gain-of-function mutants (R2456H and R2488Q), we highlight altered expression of a few genes/proteins involved in iron metabolism. Mutant cells showed increased intracellular Ca 2+ compared to WT, which was correlated to increased phosphorylation of ERK1/2, inhibition of the BMP-SMADs pathway, and suppression of HAMP transcription. Moreover, the HuH7 cells, treated with PD0325901, a potent inhibitor of ERK1/2 phosphorylation, reduced the phosphorylation of ERK1/2 with the consequent increased phosphorylation of SMAD1/5/8, confirming the link between the two pathways. Another "proof of concept" for the mechanism that links PIEZO1 to HAMP regulation was obtained by mimicking PIEZO1 activation by cell Ca 2+ overload, by the Ca 2+ ionophore A23187. There was strong down-regulation of HAMP gene expression after this Ca 2+ overload. Finally, the inhibition of PIEZO1 by GsMTx4 leads to phenotype rescue. This is the first demonstration of a direct link between PIEZO1 and iron metabolism, which defines the channel as a new hepatic iron metabolism regulator and as a possible therapeutic target of iron overload in DHS and other iron-loading anemias., (© 2019 Wiley Periodicals, Inc.)

Hereditary xerocytosis (HX) is a rare, autosomal dominant congenital hemolytic anemia (CHA) characterized by erythrocyte dehydration with presentation of various degrees of hemolytic anemia. HX is often misdiagnosed as hereditary spherocytosis or other CHA. Here we report three cases of suspected HX and one case of HX associated with β-thalassemia. Sanger method was used for sequencing cDNA of the PIEZO1 gene. Variants were evaluated for potential pathogenicity by MutationTaster, PROVEAN, PolyPhen-2 and M-CAP software, and by molecular modeling. Four different variants in the PIEZO1 gene were found, including three substitutions (p.D669H, p.D1566G, p.T1732 M) and one deletion (p.745delQ). In addition, in the patient with the p.T1732 M variant we detected a 12-nucleotide deletion in the β-globin gene leading to a deletion of amino acids 62AHGK65. The joint presence of mutations in two different genes connected with erythrocytes markedly aggravated the presentation of the disease. Bioinformatic analysis and molecular modeling strongly indicated likely deleterious effects of all four PIEZO1 variants, but co-segregation analysis showed that the p.D1566G substitution is in fact non-pathogenic. Identification of causative mutations should improve the diagnosis and management of HX and provide a new insight into the molecular basis of this complex red blood cell abnormality., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hereditary xerocytosis is a rare disorder caused by defects of red blood cell permeability that are characterized by hemolytic anemia of variable degree and iron overload. Diagnosis is usually late and confused with other hemolytic anemias, which can lead to procedural indications, such as splenectomy, contraindicated in these patients. We report the clinical, haematological, and molecular characteristics of two patients from two unrelated families affected by hereditary xerocytosis. Both patients had dehydrated erythrocytes with a high concentration of mean corpuscular hemoglobin, non-pathognomonic smears, markers of hemolysis and a resistant osmotic fragility curve. The diagnosis was confirmed by the sequencing of the PIEZO gene. We emphasize the importance of recognizing the cause of hemolytic anemia to give an accurate therapeutic approach and give adequate genetic counseling., Competing Interests: The authors report no conflicts of interest in this work., (Sociedad Argentina de Pediatría.)

Piezo1, encoded by the gene PIEZO1 , is an erythrocytic cellular membrane mechanoactivated cation channel. Mutations have been implicated in erythrocyte volume disorders (EVDs)-especially hereditary xerocytosis (HX)/dehydrated stomatocytosis (DHS). We identified three patients, all with novel PIEZO1 mutations, but only one displaying the HX/DHS phenotype. Retrospective review of three cases. Osmotic gradient red cell deformability (Osmoscan) was assessed via the Technicon Ektacytometer. Red cell band 3 content was estimated using Eosin-5'-Maleimide staining. Patient 1 was evaluated for polycythemia. Osmoscans suggested mild spherocytosis; a novel PIEZO1 mutation (p.Thr1589Ile, exon 35) was identified, causing mild erythrocytosis without hemolytic anemia. Patient 2 was evaluated for macrocytosis/reticulocytosis, normal-to-high hemoglobin, and indirect hyperbilirubinemia. Osmoscans suggested increased cellular hydration; a second novel PIEZO1 mutation (p.Arg1728Cys, exon 37) was identified, resulting in overhydrated stomatocytosis with well-compensated hemolysis. Patient 3 was evaluated for indirect hyperbilirubinemia only. Osmoscans suggested dehydrated stomatocytosis (DHS, xerocytosis); a third novel PIEZO1 mutation (p.Arg2279Cys, exon 47) was identified. All three patients' blood smears demonstrated stomatocytes and spherocytes. EVDs may be underdiagnosed due to the lack of "expected" anemia in a hemolytic disorder; two of three patients had high hemoglobin and red cell counts and one had high normal values for both parameters and the presence of stomatocytes/dehydrated cells lead to identification of causative PIEZO1 mutations. PIEZO1 -associated EVDs may be more common than previously suspected and should be included in the diagnostic algorithms for mild erythrocytosis/unexplained jaundice.

We describe the clinical, hematologic and genetic characteristics of a retrospective series of 126 subjects from 64 families with hereditary xerocytosis. Twelve patients from six families carried a KCNN4 mutation, five had the recurrent p.Arg352His mutation and one had a new deletion at the exon 7-intron 7 junction. Forty-nine families carried a PIEZO1 mutation, which was a known recurrent mutation in only one-third of the cases and private sequence variation in others; 12 new probably pathogenic missense mutations were identified. The two dominant features leading to diagnosis were hemolysis that persisted after splenectomy and hyperferritinemia, with an inconstant correlation with liver iron content assessed by magnetic resonance imaging. PIEZO1 -hereditary xerocytosis was characterized by compensated hemolysis in most cases, perinatal edema of heterogeneous severity in more than 20% of families and a major risk of post-splenectomy thrombotic events, including a high frequency of portal thrombosis. In KCNN4 -related disease, the main symptoms were more severe anemia, hemolysis and iron overload, with no clear sign of red cell dehydration; therefore, this disorder would be better described as a 'Gardos channelopathy'. These data on the largest series to date indicate that PIEZO1 -hereditary xerocytosis and Gardos channelopathy are not the same disease although they share hemolysis, a high rate of iron overload and inefficient splenectomy. They demonstrate the high variability in clinical expression as well as genetic bases of PIEZO1 -hereditary xerocytosis. These results will help to improve the diagnosis of hereditary xerocytosis and to provide recommendations on the clinical management in terms of splenectomy, iron overload and pregnancy follow-up., (Copyright© 2019 Ferrata Storti Foundation.)

Mechanosensitive ion channels rely on membrane composition to transduce physical stimuli into electrical signals. The Piezo1 channel mediates mechanoelectrical transduction and regulates crucial physiological processes, including vascular architecture and remodeling, cell migration, and erythrocyte volume. The identity of the membrane components that modulate Piezo1 function remain largely unknown. Using lipid profiling analyses, we here identify dietary fatty acids that tune Piezo1 mechanical response. We find that margaric acid, a saturated fatty acid present in dairy products and fish, inhibits Piezo1 activation and polyunsaturated fatty acids (PUFAs), present in fish oils, modulate channel inactivation. Force measurements reveal that margaric acid increases membrane bending stiffness, whereas PUFAs decrease it. We use fatty acid supplementation to abrogate the phenotype of gain-of-function Piezo1 mutations causing human dehydrated hereditary stomatocytosis. Beyond Piezo1, our findings demonstrate that cell-intrinsic lipid profile and changes in the fatty acid metabolism can dictate the cell's response to mechanical cues.

Hereditary xerocytosis is thought to be a rare genetic condition characterized by red blood cell (RBC) dehydration with mild hemolysis. RBC dehydration is linked to reduced Plasmodium infection in vitro; however, the role of RBC dehydration in protection against malaria in vivo is unknown. Most cases of hereditary xerocytosis are associated with gain-of-function mutations in PIEZO1, a mechanically activated ion channel. We engineered a mouse model of hereditary xerocytosis and show that Plasmodium infection fails to cause experimental cerebral malaria in these mice due to the action of Piezo1 in RBCs and in T cells. Remarkably, we identified a novel human gain-of-function PIEZO1 allele, E756del, present in a third of the African population. RBCs from individuals carrying this allele are dehydrated and display reduced Plasmodium infection in vitro. The existence of a gain-of-function PIEZO1 at such high frequencies is surprising and suggests an association with malaria resistance., (Copyright © 2018 Elsevier Inc. All rights reserved.)

PIEZO1 is a large mechanosensitive ion channel protein. Diseases associated with PIEZO1 include autosomal recessive generalised lymphatic dysplasia of Fotiou (GLDF) and autosomal dominant dehydrated hereditary stomatocytosis with or without pseudohyperkalemia and/or perinatal oedema (DHS). The two disorders show overlapping features, fetal hydrops/perinatal oedema have been reported in both. Electrophysiological studies suggest opposite mechanisms of action: the mutations identified in GLDF patients cause a loss-of-function mechanism of disease and mutations in DHS patients cause gain of function. This raises the question: Is the pathogenic disease mechanism behind the fetal oedema the same in the two phenotypes? In this Symposium Review, we will discuss the two conditions and highlight key questions that remain to be answered. For instance, the perinatal oedema often resolves soon after birth and we are still at a loss to understand why. Are there any mechanisms which could compensate for the faulty PIEZO1 in these patients? Are there physiological changes at birth that are less reliant on the function of PIEZO1? Thus, there is a clear need for further studies into the two disorders, in order to fully understand the role of PIEZO1 in health and disease., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Mutations in PIEZO1 are the primary cause of hereditary xerocytosis, a clinically heterogeneous, dominantly inherited disorder of erythrocyte dehydration. We used next-generation sequencing-based techniques to identify PIEZO1 mutations in individuals from 9 kindreds referred with suspected hereditary xerocytosis (HX) and/or undiagnosed congenital hemolytic anemia. Mutations were primarily found in the highly conserved, COOH-terminal pore-region domain. Several mutations were novel and demonstrated ethnic specificity. We characterized these mutations using genomic-, bioinformatic-, cell biology-, and physiology-based functional assays. For these studies, we created a novel, cell-based in vivo system for study of wild-type and variant PIEZO1 membrane protein expression, trafficking, and electrophysiology in a rigorous manner. Previous reports have indicated HX-associated PIEZO1 variants exhibit a partial gain-of-function phenotype with generation of mechanically activated currents that inactivate more slowly than wild type, indicating that increased cation permeability may lead to dehydration of PIEZO1-mutant HX erythrocytes. In addition to delayed channel inactivation, we found additional alterations in mutant PIEZO1 channel kinetics, differences in response to osmotic stress, and altered membrane protein trafficking, predicting variant alleles that worsen or ameliorate erythrocyte hydration. These results extend the genetic heterogeneity observed in HX and indicate that various pathophysiologic mechanisms contribute to the HX phenotype., (© 2017 by The American Society of Hematology.)

Parpaite and Coste introduce Piezo channels and their role in mechanotransduction., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Hereditary xerocytosis (HX) or dehydrated hereditary stomatocytosis (DHS) [OMIM 194380], in which PIEZO1 gene mutation has recently been identified, is difficult to diagnose. We report here the discovery of a PIEZO1 gene mutation in a Japanese family (father, daughter, and son) who were previously diagnosed with hereditary high phosphatidylcholine hemolytic anemia (HPCHA). All of the affected family members had non-spherocytic hemolytic anemia associated with severe hemochromatosis-related diabetes mellitus. Although the causative correlation between HPCHA and PIEZO1-gene mutated HX/DHS remains to be clarified, our findings raise an important question as to whether any of the HPCHA cases previously diagnosed in Japan may have in fact been the form of hemolytic anemia known as HX/DHS with PIEZO1 gene mutation.

Piezo1 ion channels are mediators of mechanotransduction in several cell types including the vascular endothelium, renal tubular cells and erythrocytes. Gain-of-function mutations in PIEZO1 cause an autosomal dominant haemolytic anaemia in humans called dehydrated hereditary stomatocytosis. However, the phenotypic consequence of PIEZO1 loss of function in humans has not previously been documented. Here we discover a novel role of this channel in the lymphatic system. Through whole-exome sequencing, we identify biallelic mutations in PIEZO1 (a splicing variant leading to early truncation and a non-synonymous missense variant) in a pair of siblings affected with persistent lymphoedema caused by congenital lymphatic dysplasia. Analysis of patients' erythrocytes as well as studies in a heterologous system reveal greatly attenuated PIEZO1 function in affected alleles. Our results delineate a novel clinical category of PIEZO1-associated hereditary lymphoedema.

Generalized lymphatic dysplasia (GLD) is a rare form of primary lymphoedema characterized by a uniform, widespread lymphoedema affecting all segments of the body, with systemic involvement such as intestinal and/or pulmonary lymphangiectasia, pleural effusions, chylothoraces and/or pericardial effusions. This may present prenatally as non-immune hydrops. Here we report homozygous and compound heterozygous mutations in PIEZO1, resulting in an autosomal recessive form of GLD with a high incidence of non-immune hydrops fetalis and childhood onset of facial and four limb lymphoedema. Mutations in PIEZO1, which encodes a mechanically activated ion channel, have been reported with autosomal dominant dehydrated hereditary stomatocytosis and non-immune hydrops of unknown aetiology. Besides its role in red blood cells, our findings indicate that PIEZO1 is also involved in the development of lymphatic structures.

Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Piezo has recently been identified as a family of eukaryotic mechanosensitive channels composed of subunits containing over 2,000 amino acids, without recognizable sequence similarity to other channels. Here, we present the crystal structure of a large, conserved extramembrane domain located just before the last predicted transmembrane helix of C. elegans PIEZO, which adopts a topologically distinct β sandwich fold. The structure was also determined of a point mutation located on a conserved surface at the position equivalent to the human PIEZO1 mutation found in dehydrated hereditary stomatocytosis patients (M2225R). While the point mutation does not change the overall domain structure, it does alter the surface electrostatic potential that may perturb interactions with a yet-to-be-identified ligand or protein. The lack of structural similarity between this domain and any previously characterized fold, including those of eukaryotic and bacterial channels, highlights the distinctive nature of the Piezo family of eukaryotic mechanosensitive channels., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Autosomal dominant dehydrated hereditary stomatocytosis (DHSt) usually presents as a compensated hemolytic anemia with macrocytosis and abnormally shaped red blood cells (RBCs). DHSt is part of a pleiotropic syndrome that may also exhibit pseudohyperkalemia and perinatal edema. We identified PIEZO1 as the disease gene for pleiotropic DHSt in a large kindred by exome sequencing analysis within the previously mapped 16q23-q24 interval. In 26 affected individuals among 7 multigenerational DHSt families with the pleiotropic syndrome, 11 heterozygous PIEZO1 missense mutations cosegregated with disease. PIEZO1 is expressed in the plasma membranes of RBCs and its messenger RNA, and protein levels increase during in vitro erythroid differentiation of CD34(+) cells. PIEZO1 is also expressed in liver and bone marrow during human and mouse development. We suggest for the first time a correlation between a PIEZO1 mutation and perinatal edema. DHSt patient red cells with the R2456H mutation exhibit increased ion-channel activity. Functional studies of PIEZO1 mutant R2488Q expressed in Xenopus oocytes demonstrated changes in ion-channel activity consistent with the altered cation content of DHSt patient red cells. Our findings provide direct evidence that R2456H and R2488Q mutations in PIEZO1 alter mechanosensitive channel regulation, leading to increased cation transport in erythroid cells.

Familial xerocytosis (HX) in humans is an autosomal disease that causes dehydration of red blood cells resulting in hemolytic anemia which has been traced to two individual mutations in the mechanosensitive ion channel, PIEZO1. Each mutation alters channel kinetics in ways that can explain the clinical presentation. Both mutations slowed inactivation and introduced a pronounced latency for activation. A conservative substitution of lysine for arginine (R2456K) eliminated inactivation and also slowed deactivation, indicating that this mutant's loss of charge is not responsible for HX. Fitting the current vs. pressure data to Boltzmann distributions showed that the half-activation pressure, P1/2, for M2225R was similar to that of WT, whereas mutations at position 2456 were left shifted. The absolute stress sensitivity was calibrated by cotransfection and comparison with MscL, a well-characterized mechanosensitive channel from bacteria that is driven by bilayer tension. The slope sensitivity of WT and mutant human PIEZO1 (hPIEZO1) was similar to that of MscL implying that the in-plane area increased markedly, by ∼6-20 nm(2) during opening. In addition to the behavior of individual channels, groups of hPIEZO1 channels could undergo simultaneous changes in kinetics including a loss of inactivation and a long (∼200 ms), silent latency for activation. These observations suggest that hPIEZO1 exists in spatial domains whose global properties can modify channel gating. The mutations that create HX affect cation fluxes in two ways: slow inactivation increases the cation flux, and the latency decreases it. These data provide a direct link between pathology and mechanosensitive channel dysfunction in nonsensory cells.

Dehydrated hereditary stomatocytosis is a genetic condition with defective red blood cell membrane properties that causes an imbalance in intracellular cation concentrations. Recently, two missense mutations in the mechanically activated PIEZO1 (FAM38A) ion channel were associated with dehydrated hereditary stomatocytosis. However, it is not known how these mutations affect PIEZO1 function. Here, by combining linkage analysis and whole-exome sequencing in a large pedigree and Sanger sequencing in two additional kindreds and 11 unrelated dehydrated hereditary stomatocytosis cases, we identify three novel missense mutations and one recurrent duplication in PIEZO1, demonstrating that it is the major gene for dehydrated hereditary stomatocytosis. All the dehydrated hereditary stomatocytosis-associated mutations locate at C-terminal half of PIEZO1. Remarkably, we find that all PIEZO1 mutations give rise to mechanically activated currents that inactivate more slowly than wild-type currents. This gain-of-function PIEZO1 phenotype provides insight that helps to explain the increased permeability of cations in red blood cells of dehydrated hereditary stomatocytosis patients. Our findings also suggest a new role for mechanotransduction in red blood cell biology and pathophysiology.

Hereditary xerocytosis (HX, MIM 194380) is an autosomal dominant hemolytic anemia characterized by primary erythrocyte dehydration. Copy number analyses, linkage studies, and exome sequencing were used to identify novel mutations affecting PIEZO1, encoded by the FAM38A gene, in 2 multigenerational HX kindreds. Segregation analyses confirmed transmission of the PIEZO1 mutations and cosegregation with the disease phenotype in all affected persons in both kindreds. All patients were heterozygous for FAM38A mutations, except for 3 patients predicted to be homozygous by clinical and physiologic studies who were also homozygous at the DNA level. The FAM38A mutations were both in residues highly conserved across species and within members of the Piezo family of proteins. PIEZO proteins are the recently identified pore-forming subunits of channels that mediate mechanotransduction in mammalian cells. FAM38A transcripts were identified in human erythroid cell mRNA, and discovery proteomics identified PIEZO1 peptides in human erythrocyte membranes. These findings, the first report of mutation in a mammalian mechanosensory transduction channel-associated with genetic disease, suggest that PIEZO proteins play an important role in maintaining erythrocyte volume homeostasis.