TRPC6 Contributes to the Ca2+ Leak of Human Erythrocytes

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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.