Ekaterina Shumilina

TRPC6 Contributes to the Ca 2+ Leak of Human Erythrocytes

Human erythrocytes express cation channels which contribute to the background leak of Ca2+, Na+ and K+. Excessive activation of these channels upon energy depletion, osmotic shock, Cl- depletion, or oxidative stress triggers suicidal death of erythrocytes (eryptosis), characterized by cell-shrinkage and exposure of phosphatidylserine at the cell surface. Eryptotic cells are supposed to be cleared from circulating blood. The present study aimed to identify the cation channels. RT-PCR revealed mRNA encoding the non-selective cation channel TRPC6 in erythroid progenitor cells. Western blotting indicated expression of TRPC6 protein in erythrocytes from man and wildtype mice but not from TRPC6-/- mice. According to flow-cytometry, Ca2+ entry into human ghosts prepared by hemolysis in EGTA-buffered solution containing the Ca2+ indicator Fluo3/AM was inhibited by the reducing agent dithiothreitol and the erythrocyte cation channel blockers ethylisopropylamiloride and amiloride. Loading of the ghosts with antibodies against TRPC6 or TRPC3/6/7 but neither with antibodies against TRPM2 or TRPC3 nor antibodies pre-adsorbed with the immunizing peptides inhibited ghost Ca2+ entry. Moreover, free Ca2+ concentration, cell-shrinkage, and phospholipid scrambling were significantly lower in Cl--depleted TRPC6-/- erythrocytes than in wildtype mouse erythrocytes. In conclusion, human and mouse erythrocytes express TRPC6 cation channels which participate in cation leak and Ca2+-induced suicidal death.

Accelerated Clearance of Plasmodium-infected Erythrocytes in Sickle Cell Trait and Annexin-A7 Deficiency

The course of malaria does not only depend on the virulence of the parasite Plasmodium but also on properties of host erythrocytes. Here, we show that infection of erythrocytes from human sickle cell trait (HbA/S) carriers with ring stages of P. falciparum led to significantly enhanced PGE2 formation, Ca2+ permeability, annexin-A7 degradation, phosphatidylserine (PS) exposure at the cell surface, and clearance by macrophages. P. berghei-infected erythrocytes from annexin-A7-deficient (annexin-A7-/-) mice were more rapidly cleared than infected wildtype cells. Accordingly, P. berghei-infected annexin-A7-/- mice developed less parasitemia than wildtype mice. The cyclooxygenase inhibitor aspirin decreased erythrocyte PS exposure in infected annexin-A7-/- mice and abolished the differences of parasitemia and survival between the genotypes. Conversely, the PGE2-agonist sulprostone decreased parasitemia and increased survival of wild type mice. In conclusion, PS exposure on erythrocytes results in accelerated clearance of Plasmodium ring stage-infected HbA/S or annexin-A7-/- erythrocytes and thus confers partial protection against malaria in vivo.

Ion Channels Modulating Mouse Dendritic Cell Functions

Ca2+-mediated signal transduction pathways play a central regulatory role in dendritic cell (DC) responses to diverse Ags. However, the mechanisms leading to increased [Ca2+]i upon DC activation remained ill-defined. In the present study, LPS treatment (100 ng/ml) of mouse DCs resulted in a rapid increase in [Ca2+]i, which was due to Ca2+ release from intracellular stores and influx of extracellular Ca2+ across the cell membrane. In whole-cell voltage-clamp experiments, LPS-induced currents exhibited properties similar to the currents through the Ca2+ release-activated Ca2+ channels (CRAC). These currents were highly selective for Ca2+, exhibited a prominent inward rectification of the current-voltage relationship, and showed an anomalous mole fraction and a fast Ca2+-dependent inactivation. In addition, the LPS-induced increase of [Ca2+]i was sensitive to margatoxin and ICAGEN-4, both inhibitors of voltage-gated K+ (Kv) channels Kv1.3 and Kv1.5, respectively. MHC class II expression, CCL21-dependent migration, and TNF-α and IL-6 production decreased, whereas phagocytic capacity increased in LPS-stimulated DCs in the presence of both Kv channel inhibitors as well as the ICRAC inhibitor SKF-96365. Taken together, our results demonstrate that Ca2+ influx in LPS-stimulated DCs occurs via Ca2+ release-activated Ca2+ channels, is sensitive to Kv channel activity, and is in turn critically important for DC maturation and functions.

Regulation of ion channels by the serum- and glucocorticoid-inducible kinase SGK1

The ubiquitously expressed serum‐ and glucocorticoid‐inducible kinase‐1 (SGK1) is genomically regulated by cell stress (including cell shrinkage) and several hormones (including gluco‐ and mineralocorticoids). SGK1 is activated by insulin and growth factors through PI3K and 3‐phosphoinositide‐dependent kinase PDK1. SGK1 activates a wide variety of ion channels (e.g., ENaC, SCN5A, TRPV4‐6, ROMK, Kv1.3, Kv1.5, Kv4.3, KCNE1/KCNQ1, KCNQ4, ASIC1, GluR6, ClCKa/barttin, ClC2, CFTR, and Orai/STIM), which participate in the regulation of transport, hormone release, neuroexcitability, inflammation, cell proliferation, and apoptosis. SGK1‐sensitive ion channels participate in the regulation of renal Na+ retention and K+ elimination, blood pressure, gastric acid secretion, cardiac action potential, hemostasis, and neuroexcitability. A common (~3‐5% prevalence in Caucasians and ~10% in Africans) SGK1 gene variant is associated with increased blood pressure and body weight as well as increased prevalence of type II diabetes and stroke. SGK1 further contributes to the pathophysiology of allergy, peptic ulcer, fibrosing disease, ischemia, tumor growth, and neurodegeneration. The effect of SGK1 on channel activity is modest, and the channels do not require SGK1 for basic function. SGK1‐dependent ion channel regulation may thus become pathophysiologically relevant primarily after excessive (pathological) expression. Therefore, SGK1 may be considered an attractive therapeutic target despite its broad range of functions.—Lang, F., Shumilina, E. Regulation of ion channels by the serum‐ and glucocorticoid‐inducible kinase SGK1. FASEB J. 27, 3–12 (2013). www.fasebj.org

Induction of Suicidal Erythrocyte Death by Listeriolysin from Listeria monocytogenes

Listeriolysin, the secreted cytolysin of the facultative intracellular bacterium Listeria monocytogenes, is its major virulence factor. Previously, non-lytic concentrations of listeriolysin were shown to induce Ca2+-permeable nonselective cation channels in human embryonic kidney cells. In erythrocytes, Ca2+ entry is followed by activation of K+ channels resulting in K+-exit as well as by membrane scrambling resulting in phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing erythrocytes are recognized by macrophages, engulfed, degraded and thus cleared from circulating blood. Phosphatidylserine exposure is a key event of eryptosis, the suicidal death of erythrocytes. The present study utilized patch-clamp technique, Fluo3-fluorescence, and annexin V-binding in FACS analysis to determine the effect of listeriolysin on cell membrane conductance, cytosolic free Ca2+ concentration, and phosphatidylserine exposure, respectively. Within 30 minutes, exposure of human peripheral blood erythrocytes to low concentrations of listeriolysin (which were non-hemolytic for the majority of cells) induced a Ca2+-permeable cation conductance in the erythrocyte cell membrane, increased cytosolic Ca2+ concentration, and triggered annexin V-binding. Increase of extracellular K+ concentration blunted, but did not prevent, listeriolysin-induced annexin V-binding. In conclusion, listeriolysin triggers suicidal death of erythrocytes, an effect at least partially due to depletion of intracellular K+. Listeriolysin induced suicidal erythrocyte death could well contribute to the pathophysiology of L. monocytogenes infection.

Blunted IgE-Mediated Activation of Mast Cells in Mice Lacking the Ca2+-Activated K+ Channel KCa3.1

Mast cell stimulation by Ag is followed by the opening of Ca2+-activated K+ channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca2+-activated K+ channel KCa3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of KCa3.1 knockout mice (KCa3.1−/−) and their wild-type littermates (KCa3.1+/+). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcεRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in KCa3.1−/− BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca2+ ionophore ionomycin was followed by stimulation of Ca2+-activated K+ channels and cell membrane hyperpolarization in KCa3.1+/+, but not in KCa3.1−/− BMMCs. Upon Ag stimulation, Ca2+ entry but not Ca2+ release from intracellular stores was markedly impaired in KCa3.1−/− BMMCs. Similarly, Ca2+ entry upon endothelin-1 stimulation was significantly reduced in KCa3.1−/− cells. Ag-induced release of β-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in KCa3.1−/− BMMCs compared with KCa3.1+/+ BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in KCa3.1−/− cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by ∼50%) blunted in KCa3.1−/− mice. In conclusion, KCa3.1 is required for Ca2+-activated K+ channel activity and Ca2+-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.

Ion channels and cell volume in regulation of cell proliferation and apoptotic cell death.

Cell proliferation must be accompanied by increase of cell volume and apoptosis is typically paralleled by cell shrinkage. Moreover, profound osmotic cell shrinkage may trigger apoptosis. In isotonic environment cell volume changes require the respective alterations of transport across the cell membrane. Cell proliferation is typically paralleled by activation of K(+) channels, which is required for the maintenance of the cell membrane potential, a critical determinant of Ca(2+) entry through Ca(2+) channels. The Ca(2+) entry leads to oscillations of cytosolic Ca(2+) activity which is followed by activation of Ca(2+) dependent transcription factors and by depolymerization of the actin filament network. The latter disinhibits the Na(+) H(+) exchanger and Na(+) , K(+) , 2Cl(-)cotransport thus leading to cell swelling. At some point transient activation of Cl(-) channels is required leading to transient decrease of cell volume. Apoptosis is typically paralleled by sustained activation of Cl(-) channels leading to Cl(-) , HCO-(3) and osmolyte exit. The subsequent cell shrinkage and cytosolic acidification are not counter-regulated by activation of the Na(+) /H(+) exchanger, which is inhibited and eventually degraded during apoptosis. At a later stage K(+) exit through K(+) channels decreases intracellular K(+) concentration and facilitates cell shrinkage. Sustained or excessive increase of Ca(+) triggers apoptotic cell death, typically paralleled by cell shrinkage due to activation of Ca(2+) sensitive K(+) channels. Cellular K(+) loss and cell shrinkage are supportive but not required for the induction of apoptosis. On the other hand, several studies point to a critical role of K(+) -channel inhibition in the initiation of apoptosis. Thus, alterations of K(+) channel and Ca(2+) channel activities may participate in the triggering of both, cell proliferation and apoptosis. The impact of those channels depends on magnitude and temporal organization of channel activation and on the activity of further signaling mechanisms. Accordingly, the same ion channel blockers may interfere with both, cell proliferation and apoptosis depending on cell type, regulatory environment and condition of the cell.

Potassium and Sodium Balance in U937 Cells During Apoptosis With and Without Cell Shrinkage

Staurosporine (STS) and etoposide (Eto) induced apoptosis of the human histiocytic lymphoma cells U937 were studied to determine the role of monovalent ions in apoptotic cell shrinkage. Cell shrinkage, defined as cell dehydration, was assayed by measurement of buoyant density of cells in continuous Percoll gradient. The K+ and Na+ content in cells of different density fractions was estimated by flame emission analysis. Apoptosis was evaluated by confocal microscopy and flow cytometry of acridine orange stained cells, by flow DNA cytometry and by effector caspase activity. Apoptosis of U937 cells induced by 1 µM STS for 4 h was found to be paralleled by an increase in buoyant density indicating cell shrinkage. An increase in density was accompanied by a decrease in K+ content (from 1.1 to 0.78 mmol/g protein), which exceeded the increase in Na+ content (from 0.30 to 0.34 mmol/g) and resulted in a significant decrease of the total K+ and Na+ content (from 1.4 to 1.1 mmol/g). In contrast to STS, 50 µM Eto for 4 h or 0.8-8 µM Eto for 18-24 h induced apoptosis without triggering cell shrinkage. During apoptosis of U937 cells induced by Eto the intracellular K+/Na+ ratio decreased like in the cells treated with STS, but the total K+ and Na+ content remained virtually the same due to a decrease in K+ content being nearly the same as an increase in Na+ content. Apoptotic cell dehydration correlated with the shift of the total cellular K+ and Na+ content. There was no statistically significant decrease in K+ concentration per cell water during apoptosis induced by either Eto (by 13.5%) or STS (by 8%), whereas increase in Na+ concentration per cell water was statistically significant (by 27% and 47%, respectively). The data show that apoptosis can occur without cell shrinkage-dehydration, that apoptosis with shrinkage is mostly due to a decrease in cellular K+ content, and that this decrease is not accompanied by a significant decrease of K+ concentration in cell water.

Stimulation of Ca2+-channel Orai1/STIM1 by serum- and glucocorticoid-inducible kinase 1 (SGK1)

Ca2+ signaling includes store‐operated Ca2+ entry (SOCE) following depletion of endoplas‐mic reticulum (ER) Ca2+ stores. On store depletion, the ER Ca2+ sensor STIM1 activates Orai1, the pore‐forming unit of Ca2+‐release‐activated Ca2+ (CRAC) channels. Here, we show that Orai1 is regulated by serum‐ and glucocorticoid‐inducible kinase 1 (SGK1), a growth factor‐regulated kinase. Membrane Orai1 protein abundance, /CRAC, and SOCE in human embryonic kidney (HEK293) cells stably expressing Orai1 and transfected with STIM1 were each significantly enhanced by coexpression of constitutively active SGK1 (by+81, +378, and+136%, respectively) but not by inactive K12 NSGK1. Coexpression of the ubiquitin ligase Nedd4‐2, an established negatively regulated SGK1 target, down‐regulated SOCE (by –48%) and /CRAc (by –60%), an effect reversed by expression of SGK1 (by +175 and +173%, respectively). Orai1 protein abundance and SOCE were significantly lower in mast cells from SGK1‐knockout (sgk1) mice (by –37% and –52%, respectively) than in mast cells from wild‐type (sgk1+/+) littermates. Activation of SOCE by sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase‐inhibitor thapsi‐gargin (2 μΜ) stimulated migration, an effect significantly higher (by +306%) in SGKl‐expressing than in SGKl‐expressing HEK293 cells, and also significantly higher (by +108%) in sgk1+/+ than in sgk1−/− mast cells. SGK1 is thus a novel key player in the regulation of SOCE.—Eylenstein, A., Geh‐ring, E.‐M., Heise, N., Shumilina, E., Schmidt, S., Szteyn, K., Münzer, P., Nurbaeva, M. K., Eichenmüller, M., Tyan, L., Regel, I., Föller, M., Kuhl, D., Soboloff, J., Penner, R., Lang, R. Stimulation of Ca2+‐channel Orai1/STIM1 by serum‐ and glucocorticoid‐inducible kinase 1 (SGK1). FASEB J. 25, 2012‐2021 (2011). www.fasebj.org

Transcription Factor NF-κB Regulates Expression of Pore-forming Ca2+ Channel Unit, Orai1, and Its Activator, STIM1, to Control Ca2+ Entry and Affect Cellular Functions

The serum and glucocorticoid-inducible kinase SGK1 increases the activity of Orai1, the pore forming unit of store-operated Ca2+ entry, and thus influences Ca2+-dependent cellular functions such as migration. SGK1 further regulates transcription factor nuclear factor κB (NF-κB). This study explored whether SGK1 influences transcription of Orai1 and/or STIM1, the Orai1-activating Ca2+ sensor. Orai1 and STIM1 transcript levels were decreased in mast cells from SGK1 knock-out mice and increased in HEK293 cells transfected with active S422DSGK1 but not with inactive K127NSGK1 or in S422DSGK1-transfected cells treated with the NF-κB inhibitor Wogonin (100 μm). Treatment with the stem cell factor enhanced transcript levels of STIM1 and Orai1 in sgk1+/+ but not in sgk1−/− mast cells and not in sgk1+/+ cells treated with Wogonin. Orai1 and STIM1 transcript levels were further increased in sgk1+/+ and sgk1−/− mast cells by transfection with active NF-κB subunit p65 as well as in HEK293 cells by transfection with NF-κB subunits p65/p50 or p65/p52. They were decreased by silencing of NF-κB subunits p65, p50, or p52 or by NF-κB inhibitor Wogonin (100 μm). Luciferase assay and chromatin immunoprecipitation defined NF-κB-binding sites in promoter regions accounting for NF-κB sensitive genomic regulation of STIM1 and Orai1. Store-operated Ca2+ entry was similarly increased by overexpression of p65/p50 or p65/p52 and decreased by treatment with Wogonin. Transfection of HEK293 cells with p65/p50 or p65/p52 further augmented migration. The present observations reveal powerful genomic regulation of Orai1/STIM1 by SGK1-dependent NF-κB signaling.