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

Dina A. Andrews, Lu Yang, Philip S. Low

Research output: Contribution to journalArticle

87 Scopus citations

Abstract

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 Cav2.1 channels. To confirm that a Cav2.1-like calcium channel exists in the mature erythrocyte membrane, RBC membrane preparations were immunoblotted with antiserum against the α1A subunit of the channel. A polypeptide of the expected molecular weight (190 kDa) was visualized. These studies indicate that an ω-agatoxin-TK-sensitive, Cav2.1-like calcium permeability pathway is present in the RBC membrane and that it may function under the control of kinases and phosphatases.

Original languageEnglish (US)
Pages (from-to)3392-3399
Number of pages8
JournalBlood
Volume100
Issue number9
DOIs
StatePublished - Nov 1 2002

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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