H2O2 activates red blood cell K-Cl cotransport via stimulation of a phosphatase

K-Cl cotransport is involved in volume regulation in a number of cell types. Cell swelling stimulates K-Cl cotransport, probably by inhibition of a volume-sensitive kinase. K-Cl cotransport can also be activated by oxidants and thiol reagents. We investigated the effect of H2O2 on K-Cl cotransport of LK sheep red blood cells in an attempt to identify the target of oxidants. H2O2 stimulated K-Cl cotransport. The stimulation was virtually abolished by subsequent incubation with calyculin, a protein phosphatase inhibitor. This suggests that H2O2 stimulates a calyculin-sensitive phosphatase and activates K-Cl cotransport by causing a decrease in phosphorylation of the transporter or a regulatory protein. The thiol reagent N-ethylmaleimide, which stimulates K-Cl cotransport, did not stimulate cotransport further in cells with cotransport activated by staurosporine but did stimulate cotransport further in cells with cotransport activated by H2O2. These results suggest that there are at least two distinct phosphorylation sites on the transporter or a regulator. The results also suggest that the phosphatase is associated with the membrane.