Universität Tübingen
TRPC channels in erythrocytes: Role for basal Ca2+ leak and suicidal cell death
Abstract
Prostaglandin-E2 (PGE2) is known to trigger suicidal death of nucleated cells and enucleated erythrocytes (apoptosis). In erythrocytes, Cl- depletion, energy depletion, osmotic shock, or oxidative stress also induce suicidal cell death via the formation of PGE2. The triggering of erythrocyte “apoptosis” (eryptosis) involves the activation of nonselective cation channels leading to Ca2+ entry followed by cell shrinkage and triggering of Ca2+ sensitive cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. As a cell model for erythrocytes, K562 human leukaemia cells were chosen due to their erythroid features. Experiments were performed in K562 cells to explore whether PGE2 induces apoptosis of nucleated cells similarly through cation channel activation and to possibly disclose the molecular identity of the cation channels involved. To this end, Ca2+ activity was estimated from fluorescence of the Ca2+-sensitive fluorescence dye Fluo3, mitochondrial potential from DePsipher fluorescence, phosphatidylserine exposure from annexin V binding, caspase activation from caspAce fluorescence, cell volume from FACS forward scatter, and DNA fragmentation utilizing a photometric enzyme immunoassay. Stimulation of K562 cells with PGE2 (50 µM) increased cytosolic Ca2+ activity, decreased forward scatter, depolarized the mitochondrial potential, increased annexin V binding, led to caspase activation, and resulted in DNA fragmentation. Gene silencing of the Ca2+-permeable transient receptor potential cation channel TRPC7 significantly blunted PGE2-induced triggering of PS exposure and DNA fragmentation. In conclusion, K562 cells express Ca2+-permeable TRPC7 channels which are activated by PGE2 and participate in the triggering of apoptosis. The results in nucleated K562 cells suggest the investigation of the involvement of TRPC channels in the triggering of eryptosis. RT-PCR revealed that erythroid progenitor cells differentiated from human peripheral blood expressed messenger RNA encoding for the non-selective cation channel TRPC6. Western blotting indicated expression of TRPC6 protein in erythrocyte membranes from man and wild type mice but not from TRPC6 deficient mice (TRPC6-/-). According to flow-cytometry, Ca2+ entry into human erythrocyte ghosts prepared by hemolysis in EGTA-buffered solution containing Fluo3 was inhibited by the reducing agent dithiothreitol (1 mM) and the erythrocyte cation channel blockers ethylisopropylamiloride (50 µM) and amiloride (1 mM). Loading of the ghosts with antibodies against TRPC6 or TRPC3/6/7 but neither with antibodies against TRPM2 or TRPC3 nor with antibodies pre-adsorbed with the respective immunizing peptides inhibited human ghost Ca2+ entry. Steady state Ca2+ activity as well as Cl- sensitive Ca2+ entry, cell shrinkage, and phospholipid scrambling were significantly blunted in erythrocytes from TRPC6-/- mice as compared to erythrocytes from their wildtype littermates. In conclusion, human and mouse erythrocytes express TRPC6 non-selective cation channels which participate in both, constitutive cation leak and Ca2+ induced suicidal death.
Author and committee
dc:creator, dc:contributor.*- Author
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- Föller, Michael
Identifiers
dc:identifier.*- Identifier
- hdl:10900/45137