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ZUSAMMENFASSUNGNeozytolyse bezeichnet die bevorzugte Zerstörung von jungen roten Blutzellen (Neozyten), wenn zu viele Erythrozyten vorhanden sind. Das kommt in der Raumfahrt oder beim Abstieg nach langem Höhenaufenthalt vor. Mit dem Ziel, die Existenz von Neozytolyse erstmals zu beweisen, haben wir in der Forschungsstation Jungfraujoch (3450 Hm) eine prospektive Studie durchgeführt. Neu gebildete Erythrozyten von 12 männlichen Probanden wurden durch Einnahme von nichtradioaktivem Glyzin mit seltenen Isotopen markiert: in Heidelberg (110 Hm) und während eines 19-tägigen Aufenthalts (3450 Hm). Die Eliminationsdynamik für Erythrozyten, die in Normoxie und in großer Höhe produziert wurden, unterschied sich nicht. Zusammen mit Änderungen der Retikulozytenzahlen konnte damit das Konzept der Neozytolyse nicht bestätigt werden.

Aims Total haemoglobin mass (tot-Hb) increases during high-altitude acclimatization. Normalization of tot-Hb upon descent is thought to occur via neocytolysis, the selective destruction of newly formed erythrocytes. Because convincing experimental proof of neocytolysis is lacking, we performed a prospective study on erythrocyte survival after a stay at the Jungfraujoch Research Station (JFJRS; 3450 m). Methods Newly formed erythrocytes of 12 male subjects (mean age 23.3 years) were age cohort labelled in normoxia (110 m) and during a 19-day high-altitude sojourn by ingestion of 13 C2- and 15 N-labelled glycine respectively. Elimination dynamics for erythrocytes produced in normoxia and at high altitude were measured by isotope ratio mass spectrometry of haem, by determining tot-Hb, reticulocyte counts, erythrocyte membrane protein 4.1a/4.1b ratio and by mathematical modelling. Results Tot-Hb increased by 4.7% ± 2.7% at high altitude and returned to pre-altitude values within 11 days after descent. Elimination of 13 C- (normoxia) and 15 N- (high altitude) labelled erythrocytes was not different. Erythropoietin levels and counts of CD71-positive reticulocytes decreased rapidly after descent. The band 4.1a/4.1b ratio decreased at altitude and remained low for 3-4 days after descent and normalized slowly. There was no indication of haemolysis. Conclusion We confirm a rapid normalization of tot-Hb upon descent. Based on the lack of accelerated removal of age cohorts of erythrocytes labelled at high altitude, on patterns of changes in reticulocyte counts and of the band 4.1a/4.1b ratio and on modelling, this decrease did not occur via neocytolysis, but by a reduced rate of erythropoiesis along with normal clearance of senescent erythrocytes.

Hereditary hemolytic anemias are genetic disorders that affect the shape and density of red blood cells. Genetic tests currently used to diagnose such anemias are expensive and unavailable in the majority of clinical labs. Here, we propose a method for identifying hereditary hemolytic anemias based on a standard biochemistry method, called Percoll gradient, obtained by centrifuging a patient's blood. Our hybrid approach consists on using spatial data-driven features, extracted with a convolutional neural network and spectral handcrafted features obtained from fast Fourier transform. We compare late and early feature fusion with AlexNet and VGG16 architectures. AlexNet with late fusion of spectral features performs better compared to other approaches. We achieved an average F1-score of 88% on different classes suggesting the possibility of diagnosing of hereditary hemolytic anemias from Percoll gradients. Finally, we utilize Grad-CAM to explore the spatial features used for classification., Comment: Accepted for publication at the 2021 IEEE International Symposium on Biomedical Imaging (ISBI 2021)

Key Points We show that the novel KCNN4 variant p.S314P is a gain-of-function mutation but is less severe than the previously reported p.R352H variant. The clinical heterogeneity, blurred symptoms, and absence of specific diagnostic markers make the diagnosis of Gardos channelopathy challenging.

Ca2+ is a universal signalling molecule involved in regulating cell cycle and fate, metabolism and structural integrity, motility and volume. Like other cells, red blood cells (RBCs) rely on Ca2+ dependent signalling during differentiation from precursor cells. Intracellular Ca2+ levels in the circulating human RBCs take part not only in controlling biophysical properties such as membrane composition, volume and rheological properties, but also physiological parameters such as metabolic activity, redox state and cell clearance. Extremely low basal permeability of the human RBC membrane to Ca2+ and a powerful Ca2+ pump maintains intracellular free Ca2+ levels between 30 and 60 nM, whereas blood plasma Ca2+ is approximately 1.8 mM. Thus, activation of Ca2+ uptake has an impressive impact on multiple processes in the cells rendering Ca2+ a master regulator in RBCs. Malfunction of Ca2+ transporters in human RBCs leads to excessive accumulation of Ca2+ within the cells. This is associated with a number of pathological states including sickle cell disease, thalassemia, phosphofructokinase deficiency and other forms of hereditary anaemia. Continuous progress in unravelling the molecular nature of Ca2+ transport pathways allows harnessing Ca2+ uptake, avoiding premature RBC clearance and thrombotic complications. This review summarizes our current knowledge of Ca2+ signalling in RBCs emphasizing the importance of this inorganic cation in RBC function and survival.

Red blood cells are highly deformable and present in various shapes. In blood cell disorders, only a subset of all cells is morphologically altered and relevant for the diagnosis. However, manually labeling of all cells is laborious, complicated and introduces inter-expert variability. We propose an attention based multiple instance learning method to classify blood samples of patients suffering from blood cell disorders. Cells are detected using an R-CNN architecture. With the features extracted for each cell, a multiple instance learning method classifies patient samples into one out of four blood cell disorders. The attention mechanism provides a measure of the contribution of each cell to the overall classification and significantly improves the networks classification accuracy as well as its interpretability for the medical expert.

This study focuses on characterization of the cardiac N-methyl D-aspartate receptors (NMDARs) as a target for endogenous and synthetic agonists and antagonists. Using isolated perfused rat hearts, we have shown that intracoronary administration of the NMDAR agonists and antagonists has a pronounced effect on autonomous heart function. Perfusion of rat hearts with autologous blood supplemented with NMDAR agonists was associated with induction of tachycardia, sinus arrhythmia, and ischemia occurring within physiological plasma concentration range for glutamate and glycine. Intracoronary administration of the NMDAR antagonists exerted an antiarrhythmic effect and resulted in bradycardia and improvement of capillary perfusion. Action of antagonists eliprodil, Ro25-6981, memantine, ketamine, and MK-801 on autonomous heart function diverged strikingly from that of L-type Ca channel blockers. Cardiac NMDAR subunit composition differed from that of neuronal receptors and was age specific and chamber specific. Transcripts of the GluN3A and GluN2D were found in all heart chambers, whereas expression of GluN1 and GluN2A and 2C was restricted to the atria. Expression of the GluN2B protein in ventricles increased markedly with age of the animals. The obtained data reveal that NMDARs are expressed in rat heart contributing to the autonomic heart rate regulation and the function of the cardiac conduction system.

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The recent success of deep learning approaches relies partly on large amounts of well annotated training data. For natural images object annotation is easy and cheap. For biomedical images however, annotation crucially depends on the availability of a trained expert whose time is typically expensive and scarce. To ensure efficient annotation, only the most relevant objects should be presented to the expert. Currently, no approach exists that allows to select those for a multiclass detection problem. Here, we present an active learning framework that identifies the most relevant samples from a large set of not annotated data for further expert annotation. Applied to brightfield images of red blood cells with seven subtypes, we train a faster R-CNN for single cell identification and classification, calculate a novel confidence score using dropout variational inference and select relevant images for annotation based on (i) the confidence of the single cell detection and (ii) the rareness of the classes contained in the image. We show that our approach leads to a drastic increase of prediction accuracy with already few annotated images. Our original approach improves classification of red blood cell subtypes and speeds up the annotation. This important step in diagnosing blood diseases will profit from our framework as well as many other clinical challenges that suffer from the lack of annotated training data.

Abundance and activity of N-methyl-D-aspartate (NMDA) in circulating red blood cells contributes to the maintenance of intracellular Ca

OBJECTIVES A severe donor organ shortage leads to the death of a substantial number of patients who are listed for transplantation. The use of hearts from donors after circulatory death could significantly expand the donor organ pool, but due to concerns about their viability, these are currently not used for transplantation. We propose short-term ex vivo normothermic machine perfusion (MP) to improve the viability of these ischaemic donor hearts. METHODS Hearts from male Lewis rats were subjected to 25 min of global in situ warm ischaemia (WI) (37°C), explanted, reconditioned for 60 min with normothermic (37°C) MP with diluted autologous blood and then stored for 4 h at 0-4°C in Custodiol cold preservation solution. Fresh and ischaemic hearts stored for 4 h in Custodiol were used as controls. Graft function was assessed in a blood-perfused Langendorff circuit. RESULTS During reconditioning, both the electrical activity and contractility of the ischaemic hearts recovered rapidly. Throughout the Langendorff reperfusion, the reconditioned ischaemic hearts had a higher average heart rate and better contractility compared with untreated ischaemic controls. Moreover, the reconditioned ischaemic hearts had higher tissue adenosine triphosphate levels and a trend towards improved tissue redox state. Perfusate levels of troponin T, creatine kinase and lactate dehydrogenase were not significantly lower than those of untreated ischaemic controls. The micro- and macroscopic appearance of the reconditioned ischaemic hearts were improved compared with ischaemic controls, but in both groups myocardial damage and oedema were evident. CONCLUSIONS Our results indicate that functional recovery from global WI is possible during short-term ex vivo reperfusion, allowing subsequent cold storage without compromising organ viability. We expect that once refined and validated, this approach may enable safe transplantation of hearts obtained from donation after circulatory death

The Gardos channel is a Ca2+ sensitive, K+ selective channel present in several tissues including RBCs, where it is involved in cell volume regulation. Recently, mutations at two different aminoacid residues in KCNN4 have been reported in patients with hereditary xerocytosis. We identified by whole exome sequencing a new family with two members affected by chronic hemolytic anemia carrying mutation R352H in the KCNN4 gene. No additional mutations in genes encoding for RBCs cytoskeletal, membrane or channel proteins were detected. We performed functional studies on patients’ RBCs to evaluate the effects of R352H mutation on the cellular properties and eventually on the clinical phenotype. Gardos channel hyperactivation was demonstrated in circulating erythrocytes and erythroblasts differentiated ex-vivo from peripheral CD34+ cells. Pathological alterations in the function of multiple ion transport systems were observed, suggesting the presence of compensatory effects ultimately preventing cellular dehydration in patient’s RBCs; moreover, flow cytometry and confocal fluorescence live-cell imaging showed Ca2+ overload in the RBCs of both patients and hypersensitivity of Ca2+ uptake by RBCs to swelling. Altogether these findings suggest that the ‘Gardos channelopathy’ is a complex pathology, to some extent different from the common hereditary xerocytosis.

Abundance and activity of N-methyl-D-aspartate (NMDA) in circulating red blood cells contributes to the maintenance of intracellular Ca(2+) in these cells and, by doing that, controls red cell volume, membrane stability, and O2 carrying capacity. Detection of the NMDA receptor activity in red blood cells is challenging as the number of its copies is low and shows substantial cell-to-cell heterogeneity. Receptor abundance is reliably assessed using the radiolabeled antagonist ([(3)H]MK-801) binding technique. Uptake of Ca(2+) following the NMDA receptor activation is detected in cells loaded with Ca(2+)-sensitive fluorescent dye Fluo-4 AM. Both microfluorescence live-cell imaging and flow cytometry may be used for fluorescence intensity detection. Automated patch clamp is currently used for recording of electric currents triggered by the stimulation of the NMDA receptor. These currents are mediated by the Ca(2+)-sensitive K(+) (Gardos) channels that open upon Ca(2+) uptake via the active NMDA receptor. Furthermore, K(+) flux through the Gardos channels induced by the NMDA receptor stimulation in red blood cells may be detected using unidirectional K(+)((86)Rb(+)) influx.

Summary Recently we showed that N-methyl D-aspartate receptors (NMDARs) are expressed in erythroid precursors (EPCs) and present in the circulating red blood cells (RBCs) of healthy humans, regulating intracellular Ca2+ in these cells. This study focuses on investigating the possible role of NMDARs in abnormally high Ca2+ permeability in the RBCs of patients with sickle cell disease (SCD). Protein levels of the NMDAR subunits in the EPCs of SCD patients did not differ from those in EPCs of healthy humans. However, the number and activity of the NMDARs in circulating SCD-RBCs was substantially up-regulated, being particularly high during haemolytic crises. The number of active NMDARs correlated negatively with haematocrit and haemoglobin levels in the blood of SCD patients. Calcium uptake via these non-selective cation channels was induced by RBC treatment with glycine, glutamate and homocysteine and was facilitated by de-oxygenation of SCD-RBCs. Oxidative stress and RBC dehydration followed receptor stimulation and Ca2+ uptake. Inhibition of the NMDARs with an antagonist memantine caused re-hydration and largely prevented hypoxia-induced sickling. The EPCs of SCD patients showed higher tolerance to memantine than those of healthy subjects. Consequently, NMDARs in the RBCs of SCD patients appear to be an attractive target for pharmacological intervention.

The presence of N-methyl-d-aspartate receptor (NMDAR) was previously shown in rat red blood cells (RBCs) and in a UT-7/Epo human myeloid cell line differentiating into erythroid lineage. Here we have characterized the subunit composition of the NMDAR and monitored its function during human erythropoiesis and in circulating RBCs. Expression of the NMDARs subunits was assessed in erythroid progenitors during ex vivo erythropoiesis and in circulating human RBCs using quantitative PCR and flow cytometry. Receptor activity was monitored using a radiolabeled antagonist binding assay, live imaging of Ca2+uptake, patch clamp, and monitoring of cell volume changes. The receptor tetramers in erythroid precursor cells are composed of the NR1, NR2A, 2C, 2D, NR3A, and 3B subunits of which the glycine-binding NR3A and 3B and glutamate-binding NR2C and 2D subunits prevailed. Functional receptor is required for survival of erythroid precursors. Circulating RBCs retain a low number of the receptor copies that is higher in young cells compared with mature and senescent RBC populations. In circulating RBCs the receptor activity is controlled by plasma glutamate and glycine. Modulation of the NMDAR activity in RBCs by agonists or antagonists is associated with the alterations in whole cell ion currents. Activation of the receptor results in the transient Ca2+accumulation, cell shrinkage, and alteration in the intracellular pH, which is associated with the change in hemoglobin oxygen affinity. Thus functional NMDARs are present in erythroid precursor cells and in circulating RBCs. These receptors contribute to intracellular Ca2+homeostasis and modulate oxygen delivery to peripheral tissues.

The N-methyl d-aspartate receptors (NMDARs) mediating Ca(2+) uptake upon stimulation with glutamate and glycine were recently discovered in red blood cells (RBC) of healthy humans. Activation of these receptors with agonists triggered transient Ca(2+)-dependent decrease in hemoglobin oxygen affinity in RBC suspension. The aim of this study was to assess the potential physiological relevance of this phenomenon. Two groups formed by either healthy untrained volunteers or endurance athletes were subjected to a stepwise incremental cycling test to exhaustion. Plasma glutamate levels, activity of the NMDARs, and hemoglobin O2 affinity were measured in blood samples obtained before and after the exercise in both groups. Increase in plasma glutamate levels following exercise was observed in both groups. Transient Ca(2+) accumulation in response to the NMDAR stimulation with NMDA and glycine was followed by facilitated Ca(2+) extrusion from the RBC and compensatory decrease in cytosolic Ca(2+) levels. Short-term activation of the receptors triggered a transient decrease in O2 affinity of hemoglobin in both groups. These exercise-induced responses were more pronounced in athletes compared to the untrained subjects. Athletes were initially presented with lower basal intracellular Ca(2+) levels and hemoglobin oxygen affinity compared to non-trained controls. High basal plasma glutamate levels were associated with induction of hemolysis and formation of echinocytes upon stimulation with the receptor agonists. These findings suggest that glutamate release occurring during exhaustive exercise bouts may acutely facilitate O2 liberation from hemoglobin and improve oxygen delivery to the exercising muscle.

Incubation of cerebellar neurons with homocysteine or homocysteic acid at a concentration of 500 μM for 30 min increases the intracellular level of free radicals. Specific antagonists of metabotropic glutamate receptors of the groups I (AIDA) and III (MSOP) decreased the accumulation of reactive oxygen species in cells, which suggests that homocysteine and homocysteic acid can interact with these receptors. Inhibition of NO-synthase does not affect the level of free radicals, which indicates that NO• did not contribute to an increase in the level of free radicals in neurons under our conditions. Neither homocysteine nor homocysteic acid induced neuronal death; however, both compounds inhibited neuronal Na/K-ATPase. Inhibition of Na/K-ATPase was stronger in the case of activation of ionotropic glutamate receptors by NMDA as compared to simultaneous activation of both ionotropic and metabotropic glutamate receptors by homocysteine or homocysteic acid.

Prenatal hyperhomocysteinemia induced in rats by overloading of dietary methionine (1 g/kg body mass daily) results in systemic disordering in progeny related to an increase in the excitotoxic feature of NMDA-receptors in cerebellar neurons and memory suppression. Administration of carnosine (100 mg/kg body mass daily) in the diet of pregnant rats with hyperhomocysteinemia prevents both cognitive function in pups and protects cerebellar neurons from oxidative stress. The effect of carnosine is accompanied by with the restoration of superoxide dismutase in rat brain, which is decreased during hyperhomocysteinemia from 2.07 units (control) to 1.54 units.

Homocysteine (HC) and homocysteinic acid (HCA) were capable of elevating the intracellular level of calcium ions and active oxygen forms (AOF) in rat cerebellum neurons similarly to NMDA. The NMDAreceptor antagonist, MK-801, prevented the rise of both indexes induced by HC or HCA, whereas α-methylcarboxyphenylglycine (an antagonist of metabotropic glutamate receptors of group I) did not interfere with the effect HC or HCA. Chelation of intracellular calcium by 1,2-bis(2-aminophenoxy)ethen-N,N,N′,N′-tetraacetic acid (the BAPTA intracellular calcium buffer) decreased AOF generation during neuron activation by HC or HCA to the control level. NMDA, HC, and HCA induced phosphatidylserine externalization in neurons, which was one of the early stages of apoptosis induced by AOF. Induction of apoptosis by HCA was partially prevented by BAPTA, N-acetylcysteine or cyclosporine A. These facts could be the basis of the neurotoxic effects of HC and its derivatives.

In search for the molecular identity of the Ca2+ uptake pathways in red blood cells (RBCs) we have recently discovered glutamate/homocysteic acid-sensitive Ca2+ transporter which appeared to be the...

Multiple lines of evidence demonstrate that reactive oxygen species (ROS) are involved in regulation of normal cell metabolism as second messengers. Under extreme conditions, these molecules induce oxidative stress, which may stimulate (or accompany) a number of neurodegenerative processes. In the glutamatergic system, ROS levels are under control of ionotropic and metabotropic glutamate receptors, which modulate ion fluxes through the neuronal membrane. The Na+/K(+)-pump is also one of the important participants affecting stationary ROS levels through several distinct mechanisms. This review describes the involvement of the Na+/K(+)-pump in intracellular signaling mechanisms via cross-talk between the pump and glutamate receptors in cerebellum granule cells. Selective dysfunction of mGlu II receptors may also lead to abnormal protein phosphorylation (i.e., tau phosphorylation), culminating in neurodegenerative disorders (i.e., Alzheimer disease). Also, unregulated production of intracellular ROS resulting from an imbalance of ionotropic and metabotropic receptors may activate one or more protein kinases. In summary, Glu receptor dysfunction, leading to a deficit in glutamate-mediated signal transduction may represent one of the earliest stages of neurodegenerative disorders. The Na+/K(+)-pump is able to prevent over-production of intracellular ROS, thus increasing oxidative stability of neuronal cells.

We found that methionine added to the ration of pregnant rats (1 g/kg body weight) induced sustained hyperhomocysteinemia and led to the formation of sustained oxidative stress in the brain of their progeny. Newborn animals were characterized by lower body weight, SOD deficiency in the brain, increased neuronal death, and desensitization of NMDA receptors. These factors are associated with impaired cognitive capacity in the Morris test.

Red blood cell research is important for both, the clinical haematology, such as transfusion medicine or anaemia investigations, and the basic research fields like exploring general membrane physiology or rheology.Investigations of red blood cells include a wide spectrum of methodologies ranging from population measurements with a billion cells evaluated simultaneously to single-cell approaches. All methods have a potential for pitfalls, and the comparison of data achieved by different technical approaches requires a consistent set of standards.Here, we give an overview of common mistakes using the most popular methodologies in red blood cell research and how to avoid them. Additionally, we propose a number of standards that we believe will allow for data comparison between the different techniques and different labs. We consider biochemical analysis, flux measurements, flow cytometry, patch-clamp measurements and dynamic fluorescence imaging as well as emerging single-cell techniques, such as the use of optical tweezers and atomic force microscopy.

Na,K-ATPase is highly sensitive to changes in the redox state, and yet the mechanisms of its redox sensitivity remain unclear. We have explored the possible involvement of S-glutathionylation of the catalytic α subunit in redox-induced responses. For the first time, the presence of S-glutathionylated cysteine residues was shown in the α subunit in duck salt glands, rabbit kidneys, and rat myocardium. Exposure of the Na,K-ATPase to oxidized glutathione (GSSG) resulted in an increase in the number of S-glutathionylated cysteine residues. Increase in S-glutathionylation was associated with dose- and time-dependent suppression of the enzyme function up to its complete inhibition. The enzyme inhibition concurred with S-glutathionylation of the Cys-454, -458, -459, and -244. Upon binding of glutathione to these cysteines, the enzyme was unable to interact with adenine nucleotides. Inhibition of the Na,K-ATPase by GSSG did not occur in the presence of ATP at concentrations above 0.5 mm. Deglutathionylation of the α subunit catalyzed by glutaredoxin or dithiothreitol resulted in restoration of the Na,K-ATPase activity. Oxidation of regulatory cysteines made them inaccessible for glutathionylation but had no profound effect on the enzyme activity. Regulatory S-glutathionylation of the α subunit was induced in rat myocardium in response to hypoxia and was associated with oxidative stress and ATP depletion. S-Glutathionylation was followed by suppression of the Na,K-ATPase activity. The rat α2 isoform was more sensitive to GSSG than the α1 isoform. Our findings imply that regulatory S-glutathionylation of the catalytic subunit plays a key role in the redox-induced regulation of Na,K-ATPase activity.

The Gardos channel (KCNN4) is a Ca2+ sensitive, intermediate conductance, K+ selective channel abundant in several tissues including red blood cells (RBC) where it is involved in cell volume regulation (Hoffman et al. 2003, Cahalan et al. 2015). Hereditary dehydrate stomatocytosis (DHSt) is characterized by mild to moderate congenital hemolytic anemia with reticulocytosis and splenomegaly associated with RBC dehydration. Affected RBC are characterized by a nonspecific cation leak, reflected in elevated Na+ content, decreased K+, elevated MCHC and MCV, and decreased osmotic fragility. The definitive diagnosis of DHSt is made by osmotic gradient ektacytometry, which shows a leftward shift of the bell-shaped curve. Heterozygous mutations associated with delayed channel inactivation have been identified for PIEZO1 (Zarychanski et al. 2012), although there are patients lacking a mutation in this gene. Differential diagnosis in hereditary stomatocytosis is important because in DHSt patients, splenectomy is contraindicated due to increased risk of thromboembolic complications. We studied a 40 year-old Italian man, affected since infancy by chronic hemolyitc anemia (Hb 7-9 g/dL, reticulocytes 10%). The unrelated parents and three siblings were healthy with normal hematologic parameters. He was occasionally transfused until splenectomy, performed at the age of 11 years. After splenectomy Hb levels maintained around 10 g/dL, thrombotic events did not occur. Increased ferritin levels (>1000 ng/ml) and moderate iron overload required chelation therapy. Peripheral blood smear showed anysopoikilocytosis with stomatocytes (13%) echinocytes (7%) schistocytes (6%) ellyptocytes (5%) spherocytes (4%). Bone marrow evaluation revealed erythroid hyperplasia with some dyserytrhopoietc changes, in particular binucleated erythroblasts. Eosin 5 maleimide (EMA) binding test was negative; despite the high number of stomatocytes in peripheral blood, the osmotic gradient ektacytometry curve was not suggestive for hereditary xerocytosis. The proband's first daughter, born at term after an uneventful pregnancy, presented with severe anemia (Hb 6.1 g/dL) requiring RBC transfusion at birth and at 4 months; at the age of 1 year she displayed a clinical pattern similar to the one of the father, with median Hb levels of 9 g/dL and no need of transfusions. To uncover the underlying etiologies, all the family members underwent whole exome sequencing. Only one candidate gene, the KCNN4 could be identified: a heterozygous missense mutation (c.1055G>A, p.R352H) was detected as a de novo mutation in the proband, and dominantly transmitted to the daughter. The mutation falling in exon 7 involved in the calmodulin binding domain was predicted to be pathogenic by in silico analysis. Sanger sequencing confirmed the presence of the mutation. Very recently (July 2015), the same mutation in the KCNN4 gene was described by other groups (Rapetti-Mauss et al. 2015; Andolfo et al. 2015). In heterologous expression systems it could be shown that the Ca2+ sensitivity of the mutated Gardos channel was increased. We performed single cell experiments, including patch-clamp recordings in RBCs and precursor cells, Ca2+ fluorescence imaging as well as tracer flux experiments in RBCs of the above described patients and healthy controls to get a mechanistic link between the Gardos channel mutation and the cellular symptoms. Despite the presence of a single mutation in Gardos channel alone, pathological alterations in function of multiple ion transport systems were revealed during these studies. Our findings together with recent reports (Rapetti-Mauss et al. 2015; Andolfo et al. 2015; Glogowska et al. 2015) suggest that the prevalence of mutations in the Gardos channel is higher than previously thought. Therefore it is important to understand the mechanisms implicated by the mutation and to develop reliable diagnostic tools and treatment strategies. Disclosures No relevant conflicts of interest to declare.

N-methyl-d-aspartate (NMDA) receptors are ligand-gated nonselective cation channels mediating fast neuronal transmission and long-term potentiation in the central nervous system. These channels have a 10-fold higher permeability for Ca2+compared with Na+or K+and binding of the agonists (glutamate, homocysteine, homocysteic acid, NMDA) triggers Ca2+uptake. The present study demonstrates the presence of NMDA receptors in rat erythrocytes. The receptors are most abundant in both erythroid precursor cells and immature red blood cells, reticulocytes. Treatment of erythrocytes with NMDA receptor agonists leads to a rapid increase in intracellular Ca2+resulting in a transient shrinkage via Gardos channel activation. Additionally, the exposure of erythrocytes to NMDA receptor agonists causes activation of the nitric oxide (NO) synthase facilitating either NO production in l-arginine-containing medium or superoxide anion (O2·−) generation in the absence of l-arginine. Conversely, treatment with an NMDA receptor antagonist MK-80, or the removal of Ca2+from the incubation medium causes suppression of Ca2+accumulation and prevents attendant changes in cell volume and NO/O2·−production. These results suggest that the NMDA receptor activity in circulating erythrocytes is regulated by the plasma concentrations of homocysteine and homocysteic acid. Moreover, receptor hyperactivation may contribute to an increased incidence of thrombosis during hyperhomocysteinemia.

Numerous excellent reviews are available that cover calcium signalling in cardiac myocytes; however, a comprehensive review regarding red blood cell calcium signalling has not been written. Although certain effects of calcium in red blood cells have been known for more than 50 years, the means by which calcium enters the red blood cells still has not been completely elucidated. However, what has been clear for quite some time is how the calcium is transported out of the cell once it entered the cell and how the internal calcium concentration is maintained at a low concentration (in general, approximately 60 nM) via the calcium pump. The calcium pump was initially described and characterised by Schatzmann, has been further investigated and reviewed by many others and is still not completely understood.