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TRPC6 Contributes to the Ca2+ Leak of Human ErythrocytesFöller M.* · Kasinathan R.S.* · Koka S.* · Lang C.* · Shumilina E.* · Birnbaumer L.1 · Lang F.1 · Huber S.M.1
Department of Physiology, University of Tübingen,1Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC,*M.F. and R.S.K. contributed equally to this study and therefore share first-authorship Corresponding Author
Department of Physiology I, Eberhard-Karls-University Tübingen
Gmelinstrasse 5, 72076 Tübingen (Germany)
Tel. +49 7071-29-76737, Fax: +49 7071-29-3073
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.
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