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Am J Physiol Cell Physiol 273: C1206-C1214, 1997;
0363-6143/97 $5.00
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Vol. 273, Issue 4, C1206-C1214, October 1997

Effects of PKCalpha activation on Ca2+ pump and KCa channel in deoxygenated sickle cells

Hassana Fathallah1, Monique Sauvage1, Jose R. Romero2, Mitzy Canessadagger ,3, and Françoise Giraud1

1 Laboratoire des Biomembranes et Messagers Cellulaires, Unité de Recherches Associée 1116, Centre National de la Recherche Scientifique, Université Paris XI, 91405 Orsay, France; 2 Endocrine-Hypertension Division, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215; and 3 Facultad de Ciencas, Universidad de Chile, Santiago-7, Chile

We have previously shown that a pretreatment with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), reduced deoxygenation-induced K+ loss and Ca2+ uptake and prevented cell dehydration in sickle anemia red blood cells (SS cells) (H. Fathallah, E. Coezy, R.-S. De Neef, M.-D. Hardy-Dessources, and F. Giraud. Blood 86: 1999-2007, 1995). The present study explores the detailed mechanism of this PMA-induced inhibition. The main findings are, first, the detection of PKCalpha and PKCzeta in normal red blood cells and the demonstration that both isoforms are expressed at higher levels in SS cells. The alpha -isoform only is translocated to the membrane and activated by PMA and by elevation of cytosolic Ca2+. Second, PMA is demonstrated to activate Ca2+ efflux in deoxygenated SS cells by a direct stimulation of the Ca2+ pump. PMA, moreover, inhibits deoxygenation-induced, charybdotoxin-sensitive K+ efflux in SS cells. This inhibition is partly indirect and explained by the reduced deoxygenation-induced rise in cytosolic Ca2+ resulting from Ca2+ pump stimulation. However, a significant inhibition of the Ca2+-activated K+ channels (KCa channels) by PMA can also be demonstrated when the channels are activated by Ca2+ plus ionophore, under conditions in which the Ca2+ pump is operating near its maximal extrusion rate, but swamped by Ca2+ plus ionophore. The data thus suggest a PKCalpha -mediated phosphorylation both of the Ca2+ pump and of the KCa channel or an auxiliary protein.

phosphorylation; dehydration; cationic fluxes; protein kinase C alpha ; protein kinase C zeta ; calcium-activated potassium channel

dagger Deceased 1 February 1997. 




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