Phorbol ester stimulates a protein kinase C-mediated agatoxin-TK-sensitive calcium permeability pathway in human red blood cells

Calcium entry into mature erythrocytes (red blood cells; RBCs) is associated with multiple changes in cell properties. At low intracellular Ca²⁺, efflux of potassium and water predominates, leading to changes in erythrocyte rheology. At higher Ca²⁺ content, activation of kinases and phosphatases, rupture of membrane-to-skeleton bridges, stimulation of a phospholipid scramblase and phospholipase C, and induction of transglutaminase-mediated protein cross-linking are also observed. Because the physiologic relevance of these latter responses depends partially on whether Ca²⁺ entry involves a regulated channel or nonspecific leak, we explored mechanisms that initiate controlled Ca²⁺ influx. Protein kinase C (PKC) was considered a prime candidate for the pathway regulator, and phorbol-12 myristate-13 acetate (PMA), a stimulator of PKC, was examined for its influence on erythrocyte Ca²⁺. PMA was found to stimulate a rapid, dose-dependent influx of calcium, as demonstrated by the increased fluorescence of an entrapped Ca²⁺-sensitive dye, Fluo-3/AM. The PMA-induced entry was inhibited by staurosporine and the PKC-selective inhibitor chelerythrine chloride, but was activated by the phosphatase inhibitors okadaic acid and calyculin A. The PMA-promoted calcium influx was also inhibited by ω-agatoxin-TK, a calcium channel blocker specific for Ca_v2.1 channels. To confirm that a Ca_v2.1-like calcium channel exists in the mature erythrocyte membrane, RBC membrane preparations were immunoblotted with antiserum against the α_1A subunit of the channel. A polypeptide of the expected molecular weight (190 kDa) was visualized. These studies indicate that an ω-agatoxin-TK-sensitive, Ca_v2.1-like calcium permeability pathway is present in the RBC membrane and that it may function under the control of kinases and phosphatases.