Pharmacological activation of PIEZO1 in human red blood cells prevents Plasmodium falciparum invasion.

An inherited gain-of-function variant (E756del) in the mechanosensitive cationic channel PIEZO1 was shown to confer a significant protection against severe malaria. Here, we demonstrate in vitro that human red blood cell (RBC) infection by Plasmodium falciparum is prevented by the pharmacological activation of PIEZO1. Yoda1 causes an increase in intracellular calcium associated with rapid echinocytosis that inhibits RBC invasion, without affecting parasite intraerythrocytic growth, division or egress. Notably, Yoda1 treatment significantly decreases merozoite attachment and subsequent RBC deformation. Intracellular Na+/K+ imbalance is unrelated to the mechanism of protection, although delayed RBC dehydration observed in the standard parasite culture medium RPMI/albumax further enhances the resistance to malaria conferred by Yoda1. The chemically unrelated Jedi2 PIEZO1 activator similarly causes echinocytosis and RBC dehydration associated with resistance to malaria invasion. Spiky outward membrane projections are anticipated to reduce the effective surface area required for both merozoite attachment and internalization upon pharmacological activation of PIEZO1. Globally, our findings indicate that the loss of the typical biconcave discoid shape of RBCs, together with an altered optimal surface to volume ratio, induced by PIEZO1 pharmacological activation prevent efficient P. falciparum invasion.

SK2 channels set a signalling hub bolstering CAF-triggered tumourigenic processes in pancreatic cancer.

OBJECTIVE: Intercellular communication within pancreatic ductal adenocarcinoma (PDAC) dramatically contributes to metastatic processes. The underlying mechanisms are poorly understood, resulting in a lack of targeted therapy to counteract stromal-induced cancer cell aggressiveness. Here, we investigated whether ion channels, which remain understudied in cancer biology, contribute to intercellular communication in PDAC. DESIGN: We evaluated the effects of conditioned media from patient-derived cancer-associated fibroblasts (CAFs) on electrical features of pancreatic cancer cells (PCC). The molecular mechanisms were deciphered using a combination of electrophysiology, bioinformatics, molecular and biochemistry techniques in cell lines and human samples. An orthotropic mouse model where CAF and PCC were co-injected was used to evaluate tumour growth and metastasis dissemination. Pharmacological studies were carried out in the Pdx1-Cre, Ink4afl/fl LSL-KrasG12D (KICpdx1) mouse model. RESULTS: We report that the K+ channel SK2 expressed in PCC is stimulated by CAF-secreted cues (8.84 vs 2.49 pA/pF) promoting the phosphorylation of the channel through an integrin-epidermal growth factor receptor (EGFR)-AKT (Protein kinase B) axis. SK2 stimulation sets a positive feedback on the signalling pathway, increasing invasiveness in vitro (threefold) and metastasis formation in vivo. The CAF-dependent formation of the signalling hub associating SK2 and AKT requires the sigma-1 receptor chaperone. The pharmacological targeting of Sig-1R abolished CAF-induced activation of SK2, reduced tumour progression and extended the overall survival in mice (11.7 weeks vs 9.5 weeks). CONCLUSION: We establish a new paradigm in which an ion channel shifts the activation level of a signalling pathway in response to stromal cues, opening a new therapeutic window targeting the formation of ion channel-dependent signalling hubs.

Next generation sequencing (NGS) interest in deciphering erythrocyte molecular defects' association in red cell disorders: Clinical and erythrocyte phenotypes of patients with mutations inheritance in PIEZO1, Spectrin ß1, RhAG and SLC4A1.

We report here an instructive case referred at 16 months-old for exploration of hemolysis without anemia (compensated anemia with reticulocytosis). The biology tests confirmed the hemolysis with increased total and indirect bilirubin. The usual hemolysis diagnosis tests were normal (DAT, G6PD, PK, Hb electrophoresis) except cytology and ektacytometry suggesting an association of multiple red blood cell (RBC) membrane disorders. This led us to propose a molecular screening analysis using targeted-Next Generation Sequencing (t-NGS) with a capture technique on 93 genes involved in RBC and erythropoiesis defects. We identified 4 missense heterozygous allelic variations, all of them were described without any significance (VUS) in the SLC4A1, RhAG, PIEZO1 and SPTB genes. The study of the familial cosegregation and research functional tests allowed to decipher the role of at least two by two genes in the phenotype and the hemolytic disease of this young patient. Specialized t-NGS panel (or virtual exome/genome sequencing) in a disease-referent laboratory and the motivated collaboration of clinicians, biologists and scientists should be the gold standard for improving the diagnosis of the patients affected with RBC diseases or rare inherited anemias.

Piezo1 and BKCa channels in human atrial fibroblasts: Interplay and remodelling in atrial fibrillation.

AIMS: Atrial Fibrillation (AF) is an arrhythmia of increasing prevalence in the aging populations of developed countries. One of the important indicators of AF is sustained atrial dilatation, highlighting the importance of mechanical overload in the pathophysiology of AF. The mechanisms by which atrial cells, including fibroblasts, sense and react to changing mechanical forces, are not fully elucidated. Here, we characterise stretch-activated ion channels (SAC) in human atrial fibroblasts and changes in SAC- presence and activity associated with AF. METHODS AND RESULTS: Using primary cultures of human atrial fibroblasts, isolated from patients in sinus rhythm or sustained AF, we combine electrophysiological, molecular and pharmacological tools to identify SAC. Two electrophysiological SAC- signatures were detected, indicative of cation-nonselective and potassium-selective channels. Using siRNA-mediated knockdown, we identified the cation-nonselective SAC as Piezo1. Biophysical properties of the potassium-selective channel, its sensitivity to calcium, paxilline or iberiotoxin (blockers), and NS11021 (activator), indicated presence of calcium-dependent 'big potassium channels' (BKCa). In cells from AF patients, Piezo1 activity and mRNA expression levels were higher than in cells from sinus rhythm patients, while BKCa activity (but not expression) was downregulated. Both Piezo1-knockdown and removal of extracellular calcium from the patch pipette resulted in a significant reduction of BKCa current during stretch. No co-immunoprecipitation of Piezo1 and BKCa was detected. CONCLUSIONS: Human atrial fibroblasts contain at least two types of ion channels that are activated during stretch: Piezo1 and BKCa. While Piezo1 is directly stretch-activated, the increase in BKCa activity during mechanical stimulation appears to be mainly secondary to calcium influx via SAC such as Piezo1. During sustained AF, Piezo1 is increased, while BKCa activity is reduced, highlighting differential regulation of both channels. Our data support the presence and interplay of Piezo1 and BKCa in human atrial fibroblasts in the absence of physical links between the two channel proteins.

Erythroid glucose transport in health and disease.

Glucose transport is intimately linked to red blood cell physiology. Glucose is the unique energy source for these cells, and defects in glucose metabolism or transport activity are associated with impaired red blood cell morphology and deformability leading to reduced lifespan. In vertebrate erythrocytes, glucose transport is mediated by GLUT1 (in humans) or GLUT4 transporters. These proteins also account for dehydroascorbic acid (DHA) transport through erythrocyte membrane. The peculiarities of glucose transporters and the red blood cell pathologies involving GLUT1 are summarized in the present review.

A mutation in the Gardos channel is associated with hereditary xerocytosis.

The Gardos channel is a Ca(2+)-sensitive, intermediate conductance, potassium selective channel expressed in several tissues including erythrocytes and pancreas. In normal erythrocytes, it is involved in cell volume modification. Here, we report the identification of a dominantly inherited mutation in the Gardos channel in 2 unrelated families and its association with chronic hemolysis and dehydrated cells, also referred to as hereditary xerocytosis (HX). The affected individuals present chronic anemia that varies in severity. Their red cells exhibit a panel of various shape abnormalities such as elliptocytes, hemighosts, schizocytes, and very rare stomatocytic cells. The missense mutation concerns a highly conserved residue among species, located in the region interacting with Calmodulin and responsible for the channel opening and the K(+) efflux. Using 2-microelectrode experiments on Xenopus oocytes and patch-clamp electrophysiology on HEK293 cells, we demonstrated that the mutated channel exhibits a higher activity and a higher Ca(2+) sensitivity compared with the wild-type (WT) channel. The mutated channel remains sensitive to inhibition suggesting that treatment of this type of HX by a specific inhibitor of the Gardos channel could be considered. The identification of a KCNN4 mutation associated with chronic hemolysis constitutes the first report of a human disease caused by a defect of the Gardos channel.

Advances in understanding the pathogenesis of the red cell volume disorders.

Genetic defects of erythrocyte transport proteins cause disorders of red blood cell volume that are characterized by abnormal permeability to the cations Na(+) and K(+) and, consequently, by changes in red cell hydration. Clinically, these disorders are associated with chronic haemolytic anaemia of variable severity and significant co-morbidities, such as iron overload. This review provides an overview of recent insights into the molecular basis of this group of rare anaemias involving cation channels and transporters dysfunction. To date, a total of 5 different membrane proteins have been reported to be responsible for volume homeostasis alteration when mutated, 3 of them leading to overhydrated cells (AE1 [also termed SLC4A1], RHAG and GLUT1 [also termed SCL2A1) and 2 others to dehydrated cells (PIEZO1 and the Gardos Channel). These findings are not only of basic scientific interest, but also of direct clinical significance for improving diagnostic procedures and identify potential approaches for novel therapeutic strategies.

Structural model of the anion exchanger 1 (SLC4A1) and identification of transmembrane segments forming the transport site.

The anion exchanger 1 (AE1), a member of bicarbonate transporter family SLC4, mediates an electroneutral chloride/bicarbonate exchange in physiological conditions. However, some point mutations in AE1 membrane-spanning domain convert the electroneutral anion exchanger into a Na(+) and K(+) conductance or induce a cation leak in a still functional anion exchanger. The molecular determinants that govern ion movement through this transporter are still unknown. The present study was intended to identify the ion translocation pathway within AE1. In the absence of a resolutive three-dimensional structure of AE1 membrane-spanning domain, in silico modeling combined with site-directed mutagenesis experiments was done. A structural model of AE1 membrane-spanning domain is proposed, and this model is based on the structure of a uracil-proton symporter. This model was used to design cysteine-scanning mutagenesis on transmembrane (TM) segments 3 and 5. By measuring AE1 anion exchange activity or cation leak, it is proposed that there is a unique transport site comprising TM3-5 and TM8 that should function as an anion exchanger and a cation leak.

Monovalent cation leaks in human red cells caused by single amino-acid substitutions in the transport domain of the band 3 chloride-bicarbonate exchanger, AE1.

We identified 11 human pedigrees with dominantly inherited hemolytic anemias in both the hereditary stomatocytosis and spherocytosis classes. Affected individuals in these families had an increase in membrane permeability to Na and K that is particularly marked at 0 degrees C. We found that disease in these pedigrees was associated with a series of single amino-acid substitutions in the intramembrane domain of the erythrocyte band 3 anion exchanger, AE1. Anion movements were reduced in the abnormal red cells. The 'leak' cation fluxes were inhibited by SITS, dipyridamole and NS1652, chemically diverse inhibitors of band 3. Expression of the mutated genes in Xenopus laevis oocytes induced abnormal Na and K fluxes in the oocytes, and the induced Cl transport was low. These data are consistent with the suggestion that the substitutions convert the protein from an anion exchanger into an unregulated cation channel.

Stomatin-deficient cryohydrocytosis results from mutations in SLC2A1: a novel form of GLUT1 deficiency syndrome.

The hereditary stomatocytoses are a series of dominantly inherited hemolytic anemias in which the permeability of the erythrocyte membrane to monovalent cations is pathologically increased. The causative mutations for some forms of hereditary stomatocytosis have been found in the transporter protein genes, RHAG and SLC4A1. Glucose transporter 1 (glut1) deficiency syndromes (glut1DSs) result from mutations in SLC2A1, encoding glut1. Glut1 is the main glucose transporter in the mammalian blood-brain barrier, and glut1DSs are manifested by an array of neurologic symptoms. We have previously reported 2 cases of stomatin-deficient cryohydrocytosis (sdCHC), a rare form of stomatocytosis associated with a cold-induced cation leak, hemolytic anemia, and hepatosplenomegaly but also with cataracts, seizures, mental retardation, and movement disorder. We now show that sdCHC is associated with mutations in SLC2A1 that cause both loss of glucose transport and a cation leak, as shown by expression studies in Xenopus oocytes. On the basis of a 3-dimensional model of glut1, we propose potential mechanisms underlying the phenotypes of the 2 mutations found. We investigated the loss of stomatin during erythropoiesis and find this occurs during reticulocyte maturation and involves endocytosis. The molecular basis of the glut1DS, paroxysmal exercise-induced dyskinesia, and sdCHC phenotypes are compared and discussed.

Dual transport properties of anion exchanger 1: the same transmembrane segment is involved in anion exchange and in a cation leak.

Previous results suggested that specific point mutations in human anion exchanger 1 (AE1) convert the electroneutral anion exchanger into a monovalent cation conductance. In the present study, the transport site for anion exchange and for the cation leak has been studied by cysteine scanning mutagenesis and sulfhydryl reagent chemistry. Moreover, the role of some highly conserved amino acids within members of the SLC4 family to which AE1 belongs has been assessed in AE1 transport properties. The results suggest that the same transport site within the AE1 spanning domain is involved in anion exchange or in cation transport. A functioning mechanism for this transport site is proposed according to transport properties of the different studied point mutations of AE1.

Cancer Associated Fibroblast (CAF) Regulation of PDAC Parenchymal (CPC) and CSC Phenotypes Is Modulated by ECM Composition.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of all cancers, having one of the lowest five-year survival rates. One of its hallmarks is a dense desmoplastic stroma consisting in the abnormal accumulation of extracellular matrix (ECM) components, especially Collagen I. This highly fibrotic stroma embeds the bulk cancer (parenchymal) cells (CPCs), cancer stem cells (CSCs) and the main producers of the stromal reaction, the Cancer Associated Fibroblasts (CAFs). Little is known about the role of the acellular ECM in the interplay of the CAFs with the different tumor cell types in determining their phenotypic plasticity and eventual cell fate. METHODS: Here, we analyzed the role of ECM collagen I in modulating the effect of CAF-derived signals by incubating PDAC CPCs and CSCs grown on ECM mimicking early (low collagen I levels) and late (high collagen I levels) stage PDAC stroma with conditioned medium from primary cultured CAFs derived from patients with PDAC in a previously described three-dimensional (3D) organotypic model of PDAC. RESULTS: We found that CAFs (1) reduced CPC growth while favoring CSC growth independently of the ECM; (2) increased the invasive capacity of only CPCs on the ECM mimicking the early tumor; and (3) favored vasculogenic mimicry (VM) especially of the CSCs on the ECM mimicking an early tumor. CONCLUSIONS: We conclude that the CAFs and acellular stromal components interact to modulate the tumor behaviors of the PDAC CPC and CSC cell types and drive metastatic progression by stimulating the phenotypic characteristics of each tumor cell type that contribute to metastasis.

South-east Asian ovalocytosis and the cryohydrocytosis form of hereditary stomatocytosis show virtually indistinguishable cation permeability defects.

The hereditary stomatocytoses are a group of dominantly inherited conditions in which the osmotic stability of the red cell is compromised by abnormally high cation permeability. This report demonstrates the very marked similarities between the cryohydrocytosis form of hereditary stomatocytosis and the common tropical condition south-east Asian ovalocytosis (SAO). We report two patients, one showing a novel cryohydrocytosis variant (Ser762Arg in SLC4A1) and a case of SAO. Both cases showed a mild haemolytic state with some stomatocytes on the blood film, abnormal intracellular sodium and potassium levels which were made markedly abnormal by storage of blood at 0°C, increased cation 'leak' fluxes at 37°C and increased Na(+) K(+) pump activity. In both cases, the anion exchange function of the mutant band 3 was destroyed. Extensive electrophysiological studies comparing the cation leak and conductance in Xenopus laevis oocytes expressing the two mutant genes showed identical patterns of abnormality. These data are consistent with the cryohydrocytosis form of hereditary stomatocytosis and we conclude that the cation leak in SAO is indistinguishable from that in cryohydrocytosis, and that SAO should be considered to be an example of hereditary stomatocytosis.

The monovalent cation leak in overhydrated stomatocytic red blood cells results from amino acid substitutions in the Rh-associated glycoprotein.

Overhydrated hereditary stomatocytosis (OHSt) is a rare dominantly inherited hemolytic anemia characterized by a profuse membrane leak to monovalent cations. Here, we show that OHSt red cell membranes contain slightly reduced amounts of Rh-associated glycoprotein (RhAG), a putative gas channel protein. DNA analysis revealed that the OHSt patients have 1 of 2 heterozygous mutations (t182g, t194c) in RHAG that lead to substitutions of 2 highly conserved amino acids (Ile61Arg, Phe65Ser). Unexpectedly, expression of wild-type RhAG in Xenopus laevis oocytes induced a monovalent cation leak; expression of the mutant RhAG proteins induced a leak about 6 times greater than that in wild type. RhAG belongs to the ammonium transporter family of proteins that form pore-like structures. We have modeled RhAG on the homologous Nitrosomonas europaea Rh50 protein and shown that these mutations are likely to lead to an opening of the pore. Although the function of RhAG remains controversial, this first report of functional RhAG mutations supports a role for RhAG as a cation pore.

Band 3 Edmonton I, a novel mutant of the anion exchanger 1 causing spherocytosis and distal renal tubular acidosis.

dRTA (distal renal tubular acidosis) and HS (hereditary spherocytosis) are two diseases that can be caused by mutations in the gene encoding the AE1 (anion exchanger 1; Band 3). dRTA is characterized by defective urinary acidification, leading to metabolic acidosis, renal stones and failure to thrive. HS results in anaemia, which may require regular blood transfusions and splenectomy. Mutations in the gene encoding AE1 rarely cause both HS and dRTA. In the present paper, we describe a novel AE1 mutation, Band 3 Edmonton I, which causes dominant HS and recessive dRTA. The patient is a compound heterozygote with the new mutation C479W and the previously described mutation G701D. Red blood cells from the patient presented a reduced amount of AE1. Expression in a kidney cell line showed that kAE1 (kidney AE1) C479W is retained intracellularly. As kAE1 is a dimer, we performed co-expression studies and found that, in kidney cells, kAE1 C479W and G701D proteins traffic independently from each other despite their ability to form heterodimers. Therefore the patient carries one kAE1 mutant that is retained in the Golgi (G701D) and another kAE1 mutant (C479W) located in the endoplasmic reticulum of kidney cells, and is thus probably unable to reabsorb bicarbonate into the blood. We conclude that the C479W mutant is a novel trafficking mutant of AE1, which causes HS due to a decreased cell-surface AE1 protein and results in dRTA due to its intracellular retention in kidney.

Do We Store Packed Red Blood Cells under "Quasi-Diabetic" Conditions?

Red blood cell (RBC) transfusion is one of the most common therapeutic procedures in modern medicine. Although frequently lifesaving, it often has deleterious side effects. RBC quality is one of the critical factors for transfusion efficacy and safety. The role of various factors in the cells' ability to maintain their functionality during storage is widely discussed in professional literature. Thus, the extra- and intracellular factors inducing an accelerated RBC aging need to be identified and therapeutically modified. Despite the extensively studied in vivo effect of chronic hyperglycemia on RBC hemodynamic and metabolic properties, as well as on their lifespan, only limited attention has been directed at the high sugar concentration in RBCs storage media, a possible cause of damage to red blood cells. This mini-review aims to compare the biophysical and biochemical changes observed in the red blood cells during cold storage and in patients with non-insulin-dependent diabetes mellitus (NIDDM). Given the well-described corresponding RBC alterations in NIDDM and during cold storage, we may regard the stored (especially long-stored) RBCs as "quasi-diabetic". Keeping in mind that these RBC modifications may be crucial for the initial steps of microvascular pathogenesis, suitable preventive care for the transfused patients should be considered. We hope that our hypothesis will stimulate targeted experimental research to establish a relationship between a high sugar concentration in a storage medium and a deterioration in cells' functional properties during storage.

Hereditary Xerocytosis: Differential Behavior of PIEZO1 Mutations in the N-Terminal Extracellular Domain Between Red Blood Cells and HEK Cells.

Hereditary Xerocytosis, a rare hemolytic anemia, is due to gain of function mutations in PIEZO1, a non-selective cation channel activated by mechanical stress. How these PIEZO1 mutations impair channel function and alter red blood cell (RBC) physiology, is not completely understood. Here, we report the characterization of mutations in the N-terminal part of the protein (V598M, F681S and the double mutation G782S/R808Q), a part of the channel that was subject of many investigations to decipher its role in channel gating. Our data show that the electrophysiological features of these PIEZO1 mutants expressed in HEK293T cells are different from previously characterized PIEZO1 mutations that are located in the pore or at the C-terminal extracellular domain of the protein. Although RBC with PIEZO1 mutations showed a dehydrated phenotype, the activity of V598M, F681S or R808Q in response to stretch was not significantly different from the WT channels. In contrast, the G782S mutant showed larger currents compared to the WT PIEZO1. Interestingly, basal activity of all the mutated channels was not significantly altered at the opposite of what was expected according to the decreased water and cation contents of resting RBC. In addition, the features of mutant PIEZO1 expressed in HEK293 cells do not always correlate with the observation in RBC where PIEZO1 mutations induced a cation leak associated with an increased conductance. Our work emphasizes the role of the membrane environment in PIEZO1 activity and the need to characterize RBC permeability to assess pathogenicity to PIEZO1 mutants associated with erythrocyte diseases.