Littérature scientifique sur le sujet « Gárdos channel »

The Gárdos channel (KCNN4) and Piezo1 are the best-known ion channels in the red blood cell (RBC) membrane. Nevertheless, the quantitative electrophysiological behavior of RBCs and its heterogeneity are still not completely understood. Here, we use state-of-the-art biochemical methods to probe for the abundance of the channels in RBCs. Furthermore, we utilize automated patch clamp, based on planar chips, to compare the activity of the two channels in reticulocytes and mature RBCs. In addition to this characterization, we performed membrane potential measurements to demonstrate the effect of channel activity and interplay on the RBC properties. Both the Gárdos channel and Piezo1, albeit their average copy number of activatable channels per cell is in the single-digit range, can be detected through transcriptome analysis of reticulocytes. Proteomics analysis of reticulocytes and mature RBCs could only detect Piezo1 but not the Gárdos channel. Furthermore, they can be reliably measured in the whole-cell configuration of the patch clamp method. While for the Gárdos channel, the activity in terms of ion currents is higher in reticulocytes compared to mature RBCs, for Piezo1, the tendency is the opposite. While the interplay between Piezo1 and Gárdos channel cannot be followed using the patch clamp measurements, it could be proved based on membrane potential measurements in populations of intact RBCs. We discuss the Gárdos channel and Piezo1 abundance, interdependencies and interactions in the context of their proposed physiological and pathophysiological functions, which are the passing of small constrictions, e.g., in the spleen, and their active participation in blood clot formation and thrombosis.

Abstract In patients with Gárdos channelopathy (p.R352H), an increased concentration of intracellular Ca2+ was previously reported. This is a surprising finding because the Gárdos channel (KCa3.1) is a K+ channel. Here, we confirm the increased intracellular Ca2+ for patients with the KCa3.1 mutation p.S314P. Furthermore, we provide the concept of KCa3.1 activity resulting in a flickering of red blood cell (RBC) membranepotential, which activates the CaV2.1 channel allowing Ca2+ to enter the RBC. Activity of the nonselective cation channel Piezo1 modulates the aforementioned interplay in away that a closed Piezo1 is in favor of the KCa3.1-CaV2.1 interaction. In contrast, Piezo1 openings compromise the membrane potential flickering, thus limiting the activity of CaV2.1. With the compound NS309, we mimic a gain-of-function mutation of KCa3.1. Assessing the RBC Ca2+ response by Fluo-4–based flow cytometry and by measuring the membrane potential using the Macey-Bennekou-Egée method, we provide data that support the concept of the KCa3.1/CaV2.1/Piezo1 interplay as a partial explanation for an increased number of high Ca2+ RBCs. With the pharmacological inhibition of KCa3.1 (TRAM34 and Senicapoc), CaV2.1 (ω-agatoxin TK), and Piezo1 (GsMTx-4), we could project the NS309 behavior of healthy RBCs to the RBCs of Gárdos channelopathy patients.

Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.

Handbooks of physiology state that the strategy adopted by red blood cells (RBCs) to preserve cell volume is to maintain membrane permeability for cations at its minimum. However, enhanced cation permeability can be measured and observed in specific physiological and pathophysiological situations such as in vivo senescence, storage at low temperature, sickle cell anemia and many other genetic defects affecting transporters, membrane or cytoskeletal proteins. Among cation pathways, cation channels are able to dissipate rapidly the gradients that are built and maintained by the sodium and calcium pumps. These situations are very well-documented but a mechanistic understanding of complex electrophysiological events underlying ion transports is still lacking. In addition, non-selective cation (NSC) channels present in the RBC membrane have proven difficult to molecular identification and functional characterization. For instance, NSC channel activity can be elicited by Low Ionic Strength conditions (LIS): the associated change in membrane potential triggers its opening in a voltage dependent manner. But, whereas this depolarizing media produces a spectacular activation of NSC channel, Gárdos channel-evoked hyperpolarization's have been shown to induce sodium entry through a pathway thought to be conductive and termed Pcat. Using the CCCP method, which allows to follow fast changes in membrane potential, we show here (i) that hyperpolarization elicited by Gárdos channel activation triggers sodium entry through a conductive pathway, (ii) that chloride conductance inhibition unveils such conductive cationic conductance, (iii) that the use of the specific chloride conductance inhibitor NS3623 (a derivative of Neurosearch compound NS1652), at concentrations above what is needed for full anion channel block, potentiates the non-selective cation conductance. These results indicate that a non-selective cation channel is likely activated by the changes in the driving force for cations rather than a voltage dependence mechanism per se.

PIEZO1 is a mechanosensitive non-selective cation channel, present in many cell types including Red Blood Cells (RBCs). Together with the Gárdos channel, PIEZO1 forms in RBCs a tandem that participates in the rapid adjustment of the cell volume. The pharmacology allowing functional studies of the roles of PIEZO1 has only recently been developed, with Yoda1 as a widely used PIEZO1 agonist. In 2018, Yoda1 analogues were developed, as a step towards an improved understanding of PIEZO1 roles and functions. Among these, Dooku1 was the most promising antagonist of Yoda1-induced effects, without having any ability to activate PIEZO1 channels. Since then, Dooku1 has been used in various cell types to antagonize Yoda1 effects. In the present study using RBCs, Dooku1 shows an apparent IC50 on Yoda1 effects of 90.7 µM, one order of magnitude above the previously reported data on other cell types. Unexpectedly, it was able, by itself, to produce entry of calcium sufficient to trigger Gárdos channel activation. Moreover, Dooku1 evoked a rise in intracellular sodium concentrations, suggesting that it targets a non-selective cation channel. Dooku1 effects were abolished upon using GsMTx4, a known mechanosensitive channel blocker, indicating that Dooku1 likely targets PIEZO1. Our observations lead to the conclusion that Dooku1 behaves as a PIEZO1 agonist in the RBC membrane, similarly to Yoda1 but with a lower potency. Taken together, these results show that the pharmacology of PIEZO1 in RBCs must be interpreted with care especially due to the unique characteristics of RBC membrane and associated cytoskeleton.

For some molecular players in red blood cells, the functional indications and molecular evidence are discrepant. One such protein is transient potential receptor channel of canonical type 6 (TRPC6). Transcriptome analysis of reticulocytes revealed the presence of TRPC6 in mouse red blood cells and its absence in human red blood cells. We transfused TRPC6 knockout (KO) red blood cells into wild-type (WT) mice and performed functional tests; we observed the 'rescue' of TRPC6 within 10 days but slower TRPC6 'rescue' in splenectomized mice. The latter finding led us to mimic the mechanical challenge with the cantilever of an atomic force microscope (AFM) and simultaneously carry out imaging by confocal (3D) microscopy. We observed the strong interaction of red blood cells with the opposed surface in the range of 200 pN and the formation of tethers. The results of both the transfusion experiments and the atomic force spectroscopy suggest mechanically stimulated protein transfer to red blood cells as a protein source in the absence of the translational machinery. This protein transfer mechanism has the potential to be utilized in therapeutic contexts, especially for hereditary diseases involving red blood cells, such as hereditary xerocytosis or Gárdos channelopathy.

Au cours de leur vie, les érythrocytes circulent dans tout le corps pour transporter les gaz respiratoires et remplir leurs autres fonctions. Par conséquent, les érythrocytes doivent être capable de se déformer correctement pour circuler dans tous les vaisseaux, y compris les plus petits capillaires. Cette capacité est régie par un réseau complexe associant les protéines de la membrane et du cytosquelette combiné à un rapport surface-volume finement ajusté, permettant ainsi des changements de forme instantanés pour permettre un transit rapide des GR. Cela montre à quel point il est important de maintenir le volume cellulaire pour assurer 120 jours dans la circulation. Le volume cellulaire ou l'état d'hydratation est directement influencé par l'activité des transporteurs membranaires, des pompes et des canaux ioniques. La perméabilité des érythrocytes, dominée par le mouvement des anions pour des raisons physiologiques, implique que le mouvement des cations doit être maintenu aussi bas que possible pour éviter toute modification du volume cellulaire. Cependant, dans de nombreuses conditions physiopathologiques, la perméabilité aux cations est connue pour être dérégulée, conduisant à une augmentation des niveaux intracellulaires de Ca2+ et de Na+. L'objectif de ma thèse était de mieux caractériser le rôle des canaux cationiques non sélectifs (PIEZO1, TRPV2) et du canal Gárdos dans ces conditions physiopathologiques. Des expériences ont été réalisées sur des érythrocytes issus de donneurs sains ou de patients souffrant de différentes pathologies telles que la drépanocytose, la xérocytose et la stomatocytose, en utilisant des méthodes électrophysiologiques (MBE et patch-clamp), des mesures semi-quantitatives des mouvements de Ca2+ (cytométrie en flux et imagerie cellulaire) combinées à la mesure de paramètres morphométriques, ainsi que des mesures du volume cellulaire et des contenus en ions. Dans deux études indépendantes utilisant du sang de patients drépanocytaires, nous avons pu démontrer d'une part le rôle central de l'activation de PIEZO1 dans l'augmentation de la propension à la falciformation. D'autre part, nous avons démontré une sensibilité augmentée des GR drépanocytaires à la stimulation par le THC via l'activation du TRPV2. Parallèlement à ces résultats publiés, nous avons contribué à la caractérisation fonctionnelle de nombreux variants de PIEZO1 et de KCNN4, pour lesquels nous avons conçu une série d'expériences fonctionnelles afin de mieux décrire les variants génétiquement identifiés. Cette partie avec 5 variants Gárdos et 10 variants PIEZO1 apporte des éléments fonctionnels quant à la pathogénicité des mutations identifiées et souvent classées comme des variants de signification incertaine (VUS) par les algorithmes génétiques. Nous avons également pu démontrer que la molécule Dooku1, décrite dans la littérature comme un inhibiteur des effets de Yoda1, est en fait un activateur direct de PIEZO1 dans le globule rouge, contribuant à une pharmacologie plus précise de PIEZO1. En outre, nous avons mené une série d'expériences chez des patients traités à l'Alectinib (un traitement contre le cancer du poumon), pour lesquels on observe une anémie fréquente associée à une déshydratation cellulaire. L'ensemble de ces expériences a contribué à la compréhension des perméabilités aux cations dans des conditions physiologiques et physiopathologiques. Enfin, tous ces résultats remettent en évidence la particularité des GR en ce qui concerne la perméabilité cationique et les propriétés biophysiques des membranes par rapport à d'autres types de cellules et, plus important encore, lorsque des voies mécanosensibles sont impliquées dans de tels mouvements d'ions
Over their lifespan, erythrocytes circulate throughout the body to carry respiratory gases and perform their other functions. Therefore, erythrocytes must deform properly to circulate in all vessels, including the smallest of the capillaries. This ability is governed by a complex membrane-cytoskeleton network combined with a finely tuned surface-volume ratio that allows instantaneous shape changes to enable rapid RBC transit. This highlights how important it is to maintain cell volume to ensure a 120-day journey without the possibility of repair. Cell volume or hydration status is directly influenced by the activity of membrane transporters, pumps, and ion channels. The permeability of erythrocytes, which is dominated by anion movement for physiological reasons, implies that cation movement should be kept as low as possible to avoid any change in cell volume. However, in many pathophysiological conditions, cation permeabilities are known to be deregulated, leading to increased intracellular Ca2+ and Na+ levels. My thesis aimed to better characterize the role of non-selective cation (NSC) channels (PIEZO1, TRPV2), and Gárdos channel in such pathophysiological conditions. Experiments were carried out on healthy erythrocytes as well as on cells from patients suffering from different pathologies like Sickle Cell Disease (SCD), xerocytosis, and stomatocytosis, using electrophysiological methods (MBE and patch-clamp), Ca2+ movements semi-quantification (flow cytometry and live-cell imaging) combined with the measurement of morphometric parameters, and the measurements of intracellular cell volume and other ions contents. In two independent studies using blood from sickle cell patients, we were able to demonstrate from one part the central role of PIEZO1 activation in the enhancement of sickling propensity. In the other part, we demonstrated the increased sensitivity of sickle cells to THC stimulation via TRPV2 activation. Along with these published results, we have contributed to the functional characterization of many PIEZO1 and KCNN4 variants, for which we have designed a series of functional experiments to better describe the genetically identified variants. This part with 5 Gárdos and 10 PIEZO1 variants increases the knowledge about the pathogenicity of the identified mutations, often characterized as variants of uncertain significance (VUS). We were also able to demonstrate that Dooku1, a chemical compound described in the literature as an inhibitor of Yoda1's effects, is in fact, a direct activator of PIEZO1 in erythrocytes, contributing to a more accurate pharmacology of PIEZO1. Furthermore, we conducted a series of experiments in patients treated with Alectinib (a lung cancer treatment), for which frequent anemia associated with cell volume dehydration is observed. Taken together, these studies contribute to the understanding of cation permeabilities under physiological and pathophysiological conditions. Finally, all these results highlight the particularity of RBCs regarding cationic permeability and biophysical membrane properties compared to other cell types and, more importantly when mechanosensitive pathways are involved in such ion movements