title: TRPC6 contributes to the Ca(2+) leak of human erythrocytes.
abstract: Human erythrocytes express cation channels which contribute to the background leak of Ca(2+), 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, Ca(2+) entry into human ghosts prepared by hemolysis in EGTA-buffered solution containing the Ca(2+) 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 Ca(2+) entry. Moreover, free Ca(2+) 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 Ca(2+)-induced suicidal death.
authors.affiliation: Department of Physiology, University of Tubingen, Tubingen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Foller
authors.forename: Ravi S
authors.initials: RS
authors.lastname: Kasinathan
authors.forename: Saisudha
authors.initials: S
authors.lastname: Koka
authors.forename: Camelia
authors.initials: C
authors.lastname: Lang
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Shumilina
authors.forename: Lutz
authors.initials: L
authors.lastname: Birnbaumer
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
authors.forename: Stephan M
authors.initials: SM
authors.lastname: Huber
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D051379:Mice; D050052:TRPC Cation Channels / Q000378:metabolism*; D000074287:TRPC6 Cation Channel
chemical_list: D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; C492146:Trpc6 protein, mouse; D002118:Calcium