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Cell Volume Regulation in Response to Hypotonicity Is Impaired in HeLa Cells Expressing a Protein Kinase C α Mutant Lacking Kinase Activity *

Journal of Biological Chemistry, ISSN: 0021-9258, Vol: 279, Issue: 17, Page: 17681-17689
2004
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The chloride conductance (G Cl,swell ) that participates in the regulatory volume decrease process triggered by osmotic swelling in HeLa cells was impaired by removal of extracellular Ca 2+, depletion of intracellular Ca 2+ stores with thapsigargin, or by preloading the cells with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane- N,N,N ′,N ′-tetraacetic acid). Furthermore, overnight exposure to the phorbol ester tetradecanoyl phorbol acetate and acute incubation with inhibitors of the conventional protein kinase C (PKC) isoforms bisindolylmaleimide I and Gö6976 inhibited G Cl,swell. Treatment of HeLa cells with U73122, a phospholipase C inhibitor, also prevented G Cl,swell. Hypotonicity induced selective PKC α accumulation in the membrane/cytoskeleton fraction in fractionation experiments and translocation of a green fluorescent protein-PKC α fusion protein to the plasma membrane of transiently transfected HeLa cells. To further explore the role of PKCs in hypotonicity-induced G Cl,swell, HeLa clones stably expressing either a kinasedead dominant negative variant of the Ca 2+ -dependent PKC isoform α (PKC α K386R) or of the atypical PKC isoform ζ (PKCζ K275W) were generated. G Cl,swell was significantly reduced in HeLa cells expressing the dominant negative PKC α mutant but remained unaltered in cells expressing dominant negative PKCζ. These findings strongly implicate PKC α as a critical regulatory element that is required for efficient regulatory volume decrease in HeLa cells.

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