Cell volume regulation in response to hypotonicity is impaired in HeLa cells expressing a protein kinase Cα mutant lacking kinase activity

The chloride conductance (GC(l,swell)) that participates in the regulatory volume decrease process triggered by osmotic swelling in HeLa cells was impaired by removal of extracellular Ca2+, depletion of intracellular Ca2+ 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 Go6976 inhibited G(Cl,swell). Treatment of HeLa cells with U73122, a phospholipase C inhibitor, also prevented G(Cl,swell). Hypotonicity induced selective PKCalpha accumulation in the membrane/cytoskeleton fraction in fractionation experiments and translocation of a green fluorescent protein-PKCalpha 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 kinase-dead dominant negative variant of the Ca2+-dependent PKC isoform alpha (PKCalpha K386R) or of the atypical PKC isoform zeta (PKCzeta K275W) were generated. G(Cl,swell) was significantly reduced in HeLa cells expressing the dominant negative PKCalpha mutant but remained unaltered in cells expressing dominant negative PKCzeta. These findings strongly implicate PKCalpha as a critical regulatory element that is required for efficient regulatory volume decrease in HeLa cells.