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Abstract Background Sphingolipids are key molecules regulating many essential functions in eukaryotic cells and ceramide plays a central role in sphingolipid metabolism. A sphingolipid metabolism occurs in the intraerythrocytic stages of Plasmodium falciparum and is associated with essential biological processes. It constitutes an attractive and potential target for the development of new antimalarial drugs. Methods The anti-Plasmodium activity of a series of ceramide analogs containing different linkages (amide, methylene or thiourea linkages) between the fatty acid part of ceramide and the sphingoid core was investigated in culture and compared to the sphingolipid analog PPMP (d,1-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). This analog is known to inhibit the parasite sphingomyelin synthase activity and block parasite development by preventing the formation of the tubovesicular network that extends from the parasitophorous vacuole to the red cell membrane and delivers essential extracellular nutrients to the parasite. Results Analogs containing methylene linkage showed a considerably higher anti-Plasmodium activity (IC50 in the low nanomolar range) than PPMP and their counterparts with a natural amide linkage (IC50 in the micromolar range). The methylene analogs blocked irreversibly P. falciparum development leading to parasite eradication in contrast to PPMP whose effect is cytostatic. A high sensitivity of action towards the parasite was observed when compared to their effect on the human MRC-5 cell growth. The toxicity towards parasites did not correlate with the inhibition by methylene analogs of the parasite sphingomyelin synthase activity and the tubovesicular network formation, indicating that this enzyme is not their primary target. Conclusions It has been shown that ceramide analogs were potent inhibitors of P. falciparum growth in culture. Interestingly, the nature of the linkage between the fatty acid part and the sphingoid core considerably influences the antiplasmodial activity and the selectivity of analogs when compared to their cytotoxicity on mammalian cells. By comparison with their inhibitory effect on cancer cell growth, the ceramide analogs might inhibit P. falciparum growth through modulation of the endogenous ceramide level.

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.

Free Calcium (Ca 2+ ) is an important and universal signalling entity in all cells, red blood cells included. Although mature mammalian red blood cells are believed to not contain organelles as Ca 2+ stores such as the endoplasmic reticulum or mitochondria, a 20,000-fold gradient based on a intracellular Ca 2+ concentration of approximately 60 nM vs. an extracellular concentration of 1.2 mM makes Ca 2+ -permeable channels a major signalling tool of red blood cells. However, the internal Ca 2+ concentration is tightly controlled, regulated and maintained primarily by the Ca 2+ pumps PMCA1 and PMCA4. Within the last two decades it became evident that an increased intracellular Ca 2+ is associated with red blood cell clearance in the spleen and promotes red blood cell aggregability and clot formation. In contrast to this rather uncontrolled deadly Ca 2+ signals only recently it became evident, that a temporal increase in intracellular Ca 2+ can also have positive effects such as the modulation of the red blood cells O 2 binding properties or even be vital for brief transient cellular volume adaptation when passing constrictions like small capillaries or slits in the spleen. Here we give an overview of Ca 2+ channels and Ca 2+ -regulated channels in red blood cells, namely the Gárdos channel, the non-selective voltage dependent cation channel, Piezo1, the NMDA receptor, VDAC, TRPC channels, Ca V 2.1, a Ca 2+ -inhibited channel novel to red blood cells and i.a. relate these channels to the molecular unknown sickle cell disease conductance P sickle . Particular attention is given to correlation of functional measurements with molecular entities as well as the physiological and pathophysiological function of these channels. This view is in constant progress and in particular the understanding of the interaction of several ion channels in a physiological context just started. This includes on the one hand channelopathies, where a mutation of the ion channel is the direct cause of the disease, like Hereditary Xerocytosis and the Gárdos Channelopathy. On the other hand it applies to red blood cell related diseases where an altered channel activity is a secondary effect like in sickle cell disease or thalassemia. Also these secondary effects should receive medical and pharmacologic attention because they can be crucial when it comes to the life-threatening symptoms of the disease.

For many hereditary disorders, although the underlying genetic mutation may be known, the molecular mechanism leading to hemolytic anemia is still unclear and needs further investigation. Previous studies revealed an increased intracellular Ca 2+ in red blood cells (RBCs) from patients with sickle cell disease, thalassemia, or Gardos channelopathy. Therefore we analyzed RBCs' Ca 2+ content from 35 patients with different types of anemia (16 patients with hereditary spherocytosis, 11 patients with hereditary xerocytosis, 5 patients with enzymopathies, and 3 patients with hemolytic anemia of unknown cause). Intracellular Ca 2+ in RBCs was measured by fluorescence microscopy using the fluorescent Ca 2+ indicator Fluo-4 and subsequent single cell analysis. We found that in RBCs from patients with hereditary spherocytosis and hereditary xerocytosis the intracellular Ca 2+ levels were significantly increased compared to healthy control samples. For enzymopathies and hemolytic anemia of unknown cause the intracellular Ca 2+ levels in RBCs were not significantly different. These results lead us to the hypothesis that increased Ca 2+ levels in RBCs are a shared component in the mechanism causing an accelerated clearance of RBCs from the blood stream in channelopathies such as hereditary xerocytosis and in diseases involving defects of cytoskeletal components like hereditary spherocytosis. Future drug developments should benefit from targeting Ca 2+ entry mediating molecular players leading to better therapies for patients.

The phosphorylation status of red blood cell proteins is strongly altered during the infection by the malaria parasite Plasmodium falciparum. We identify the key phosphorylation events that occur in the erythrocyte membrane and cytoskeleton during infection, by a comparative analysis of global phospho-proteome screens between infected (obtained at schizont stage) and uninfected RBCs. The meta-analysis of reported mass spectrometry studies revealed a novel compendium of 495 phosphorylation sites in 182 human proteins with regulatory roles in red cell morphology and stability, with about 25% of these sites specific to infected cells. A phosphorylation motif analysis detected 7 unique motifs that were largely mapped to kinase consensus sequences of casein kinase II and of protein kinase A/protein kinase C. This analysis highlighted prominent roles for PKA/PKC involving 78 phosphorylation sites. We then compared the phosphorylation status of PKA (PKC) specific sites in adducin, dematin, Band 3 and GLUT-1 in uninfected RBC stimulated or not by cAMP to their phosphorylation status in iRBC. We showed cAMP-induced phosphorylation of adducin S59 by immunoblotting and we were able to demonstrate parasite-induced phosphorylation for adducin S726, Band 3 and GLUT-1, corroborating the protein phosphorylation status in our erythrocyte phosphorylation site compendium., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background/aims: In a variety of investigations described in the literature it was not clear to what extent the transmembrane potential red blood cells (RBCs) was changed after the cells have been transferred into low ionic strength (LIS) solutions. Another open question was to find out how fast the transmembrane potential of RBCs in LIS solution will change and which final new equilibrium value will be reached., Methods: The transmembrane potential of human and bovine RBCs was investigated using the potential-sensitive fluorescent dye DIBAC4(3) (bis(1,3-dibutylbarbituric acid) trimethine oxonol) as well as the CCCP (carbonylcyanide-m-chlorophenylhydrazone) method., Results: Under physiological conditions the transmembrane potential was about -10 mV in agreement with literature data. However, when the RBCs were transferred into an isosmotic low ionic strength medium containing sucrose the transmembrane potential increased to +73 mV and +81 mV for human and bovine RBCs, respectively. In case of human RBCs it continuously decreased reaching finally an equilibrium state of -10 mV again after 30 - 60 min. For bovine RBCs the transmembrane potential declined more slowly reaching a value of +72 mV after 30 min., Conclusions: Investigations of parameters of RBCs depending on transmembrane potential cannot be performed with human RBCs in LIS media., (Copyright © 2013 S. Karger AG, Basel.)

Previous work has shown that the transport phenotype of chicken erythrocytes changes with the age of the chicken. Here, we report changes in the transport of choline and K+ in erythrocytes from chickens at different developmental ages. The transport of choline in chicken erythrocytes was predominantly via saturable transport systems, was highest in erythrocytes from 1-day-old chickens and declined with chicken age when tested at 2 weeks of age and in mature chickens. Both Km and Vmax values for choline transport in chicken erythrocytes declined with chicken age. Similarly, the total unidirectional influx of K+ was highest in erythrocytes from 1-day-old chickens and declined with chicken age, as did ouabain-sensitive K+ influxes, which can be attributed to the Na+/K+ pump. In isotonic conditions, bumetanide-sensitive K+ influxes, which can be attributed to the Na+-K+-2Cl- cotransporter, were only measurable in erythrocytes from 1-day-old chickens. However, when stimulated by hypertonic conditions, bumetanide-sensitive K+ influxes were essentially identical in erythrocytes from 1-day- and 2-week-old chickens but decreased in erythrocytes from mature chickens. We conclude that both choline and K+ influxes decrease significantly in erythrocytes from chickens with increasing age. The changes are substantial but complex and may involve both regulation of existing transporters, and substitution or deletion of specific transporter isoforms.

Red cells infected with the human malaria parasite Plasmodium falciparum have an increased permeability to a range of small, structurally unrelated solutes via a malaria-induced pathway. We report here a similar pathway present in parasitised red cells from chickens infected with the avian malaria parasite, Plasmodium gallinaceum. Parasitised cells showed a marked increase in the rate of influx of sorbitol (76-fold) and, to a lesser degree, taurine (3-fold) when compared with red cells from uninfected chickens. Pharmacological data suggest that both sorbitol and taurine are transported via a single malaria-induced pathway, which is sensitive to inhibition by 5-nitro-2-(3-phenylpropylamino)benzoic acid (IC(50) approximately 7 microM). The malaria-induced pathway differed in its inhibition by a range of anion channel inhibitors when compared to the endogenous, volume-activated osmolyte pathway of chicken red cells. There were also differences in the selectivity of sorbitol and taurine by the two permeation routes. The data presented here are consistent with the presence of two distinct organic solute pathways in infected chicken red cells. The first is an endogenous volume-activated pathway, which is not activated by the parasite and the second is a malaria-induced pathway, similar to those that are induced by other types of malaria in other host species.

Single channel patch-clamp techniques were used to demonstrate the presence of outwardly rectifying chloride channels in the basolateral membrane of crypt cells from mouse distal colon. These channels were rarely observed in the cell-attached mode and, in the inside-out configuration, only became active after a delay and depolarizing voltage steps. Single channel conductance was 23.4 pS between -100 and -40 mV and increased to 90.2 pS between 40 and 100 mV. The channel permeability sequence for anions was: I(-) > SCN(-) > Br(-) > Cl(-) > NO(3)(-) > F(-)>> SO(4)(2-) approximately gluconate. In inside-out patches, the channel open probability was voltage dependent but insensitive to intracellular Ca(2+) concentration. In cell-attached mode, forskolin, histamine, carbachol, A-23187, and activators of protein kinase C all failed to activate the channel, and activity could not be evoked in inside-out patches by exposure to the purified catalytic subunit of cAMP-dependent protein kinase A. The channel was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, 9-anthracenecarboxylic acid, and DIDS. Stimulation of G proteins with guanosine 5'-O-(3-thiotriphosphate) decreased the channel open probability and conductance, whereas subsequent addition of guanosine 5'-O-(2-thiodiphosphate) reactivated the channel.