TRPC6 contributes to the Ca2+ 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. Copyright (c) 2008 S. Karger AG, Basel.