Altered Ca2+ Homeostasis in Red Blood Cells of Polycythemia Vera Patients Following Disturbed Organelle Sorting during Terminal Erythropoiesis

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.

The Chloride Conductance Inhibitor NS3623 Enhances the Activity of a Non-selective Cation Channel in Hyperpolarizing Conditions

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.

Mechanistic ion channel interactions in red cells of patients with Gárdos channelopathy

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.

Synthesis and biological evaluation of PET tracers designed for imaging of calcium activated potassium channel 3.1 (KCa3.1) channels in vivo

Expression of the Ca2+ activated potassium channel 3.1 (KCa3.1) channel (also known as the Gàrdos channel) is dysregulated in many tumor entities and has predictive power with respect to patient survival.

The Gardos effect drives erythrocyte senescence and leads to Lu/BCAM and CD44 adhesion molecule activation

Senescence of erythrocytes is characterized by a series of changes that precede their removal from the circulation, including loss of red cell hydration, membrane shedding, loss of deformability, phosphatidyl serine exposure, reduced membrane sialic acid content, and adhesion molecule activation. Little is known about the mechanisms that initiate these changes nor is it known whether they are interrelated. In this study, we show that Ca2+-dependent K+ efflux (the Gardos effect) drives erythrocyte senescence. We found that increased intracellular Ca2+ activates the Gardos channel, leading to shedding of glycophorin-C (GPC)–containing vesicles. This results in a loss of erythrocyte deformability but also in a marked loss of membrane sialic acid content. We found that GPC-derived sialic acid residues suppress activity of both Lutheran/basal cell adhesion molecule (Lu/BCAM) and CD44 by the formation of a complex on the erythrocyte membrane, and Gardos channel–mediated shedding of GPC results in Lu/BCAM and CD44 activation. This phenomenon was observed as erythrocytes aged and on erythrocytes that were otherwise prone to clearance from the circulation, such as sickle erythrocytes, erythrocytes stored for transfusion, or artificially dehydrated erythrocytes. These novel findings provide a unifying concept on erythrocyte senescence in health and disease through initiation of the Gardos effect.

Dehydrated Hereditary Stomatocytosis in a Multi-Generational American Family with a KCNN4 Gardos Channel Mutation

INTRODUCTION: Dehydrated hereditary stomatocytosis (DHSt) is an autosomal dominant red blood cell membrane disorder characterized by hemolytic anemia and splenomegaly. DHSt has an estimated incidence of 1:50,000 births, and the degree of anemia varies within and between families. Although transfusion support during childhood is not uncommon, continued requirement into adulthood is rare. The most frequent cause of DHSt is a gain-of-function mutation of the PIEZO1 gene, leading to delayed channel inactivation that results in a monovalent cation leak and an increase in intracellular calcium (Ca2+). Many of these patients develop recurrent thromboses post splenectomy. Other DHSt patients have mutations in KCNN4, which encodes the Gardos channel, with mutations causing increased Ca2+ sensitivity and potassium efflux. To our knowledge, 42 patients from ten families have been described with four distinct KCNN4 mutations: Arg352His, Val282Met or Val282Glu, and a 28bp deletion encompassing the exon-intron 7 junction. We report herein the eighth family with the Arg352His locus mutation. CASE REPORT: Five subjects from a single family were enrolled in this study (affected proband, unaffected husband, two affected children, and an unaffected grandchild). The proband has had hemolytic anemia since childhood. She had undergone splenectomy at age three and cholecystectomy at eight. Her anemia persisted, with a mean hemoglobin (Hgb) of 10.5g/dL and a reticulocyte count (retic ct) of 12.6%, with no need for transfusion support or iron chelation therapy (Table 1). Extensive testing revealed slightly decreased osmotic fragility and mildly elevated intracellular sodium concentration, of 19.4 mEq/L. Her daughter has mild splenomegaly and anemia, with a mean Hgb of 11.1g/dL and retic ct of 6.7%, whereas her son has more severe disease, with a mean Hgb of 9.3g/dL and a retic ct of >22%. He underwent splenectomy as a teenager for immune thrombocytopenia, and has required chelation therapy with deferasirox since age 31, when his ferritin rose to >1000ng/mL. Similar to results reported by others, splenectomy did not alter the severity of hemolysis in either the proband or her son, and neither developed thrombotic complications 57 and 7 years post splenectomy, respectively. The proband's daughter has a child with a normal Hgb, and is presumed unaffected. Stomatocytes were rarely seen on the peripheral blood smears of the proband and her children. METHODS: A clinically available 39 gene hemolytic anemia panel on the proband failed to identify the mutation underlying this disorder. We therefore performed whole exome sequencing on all five family members. We prioritized the analysis of 23 additional genes that are included in hemolytic anemia panels from two other reference laboratories and are involved in disorders of red blood cell membrane or cytoskeletal proteins of potential clinical relevance to the study population. RESULTS: A single missense mutation, Arg352His within KCNN4, was identified in all three affected individuals. This heterozygous mutation was present in the proband and her affected two children, and absent in her unaffected grandchild and husband. CONCLUSIONS: To our knowledge, this Pennsylvania family is only the eleventh described to have DHSt secondary to a KCNN4 mutation. This disorder is likely much more prevalent than reported, due to the rarity of stomatocytes on peripheral blood smears, the omission of the KCNN4 gene from hemolytic panels offered by some reference laboratories, and variable clinical presentation. KCNN4 mutations should be investigated if other causes are not identified in patients with lifelong hemolytic anemia suspected of having a red cell membrane protein or cytoskeletal disorder. Appropriate diagnosis may allow severely affected patients to be considered for treatment with the experimental Gardos channel inhibitor senicapoc. Additionally, mutational diagnosis is especially important when considering the adverse outcomes post splenectomy in PIEZO1 as compared to KCNN4 mutations. Disclosures Eyster: SPARK:Research Funding;Sanofi:Research Funding;Novo Nordisk:Research Funding;Baxalta/Shire:Research Funding.

Identification of the Anti-sickling Activity of Anogeissus leiocarpus and In Silico Investigation of Some of Its Phytochemicals

Background: The anti-sickling activity of Anogeissus leiocarpus, a plant used for managing sickle cell disease (SCD), has been previously proven. Objectives: This study investigated the anti-sickling mechanism of A. leiocarpus by probing its effects on Gardos channel (KCNN4), erythropoietin (EPO), erythropoietin receptor (EPOR), catalase (CAT), G6pD, D-type cyclins and cyclin-dependent kinase inhibitors (p21) gene expression as well as assessing in silico drug-likeness of reported compounds as EPOR agonist. Methods: A total of 18 rats (45-76 g) were selected and divided into 6 groups (n=3). The control group was given water ad libitum, standard group was given 0.1 mL/kg of Ciklavit® and experimental group was given daily oral doses of 50-100 mg/kg body weight of crude methanol extract or ethyl acetate fraction (EA-PF). Haematological parameters were analyzed while histopathological and molecular studies of kidney and bone marrow were carried out, followed by RT-PCR analysis of KCNN4, EPO, EPOR, CAT, G6pD, p21, and cyclin-dependent kinase inhibitors. Docking studies of the reported compounds were also done. Results: EA-PF had an insignificant (P>0.05) effect on haematological parameters compared to the basal group. While CAT and p21 acted in a positive feedback loop, G6pD was downregulated in the experimental groups. KCNN4 acted in a negative-feedback mechanism and the upregulation of EPO and EPOR was followed by increased reticulocytes. Kaempferol, quercetin, and catechin showed non-violation of Lipinski’s rule and high binding affinities of 6.5 kcal/mol, 6.7 kcal/mol, and 6.7 kcal/mol, respectively, for EPOR pocket compared to the co-crystallized ligand. Conclusion: Results suggest that ethyl acetate fraction of Anogeissus leiocarpus achieved a steady state level of the Gardos channel and stimulation of EPO expression via EPOR agonist.