Okadaic acid inhibition of KCl cotransport. Evidence that protein dephosphorylation is necessary for activation of transport by either cell swelling or N-ethylmaleimide

摘要

The mechanism of activation of KCl cotransport has been examined in rabbit red blood cells. Previous work has provided evidence that a net dephosphorylation is required for activation of transport by cell swelling. In the present study okadaic acid, an inhibitor of protein phosphatases, was used to test this idea in more detail. We find that okadaic acid strongly inhibits swelling-stimulated KCl cotransport. The IC50 for okadaic acid is approximately 40 nM, consistent with the involvement of type 1 protein phosphatase in transport activation. N- Ethylmaleimide (NEM) is well known to activate KCl cotransport in cells of normal volume. Okadaic acid, added before NEM, inhibits the activation of transport by NEM, indicating that a dephosphorylation is necessary for the NEM effect. Okadaic acid added after NEM inhibits transport only very slightly. After a brief exposure to NEM and rapid removal of unreacted NEM, KCl cotransport activates with a time delay that is similar to that for swelling activation. Okadaic acid causes a slight increase in the delay time. These findings are all consistent with the idea that NEM activates transport not by a direct action on the transport protein but by altering a phosphorylation- dephosphorylation cycle. The simplest hypothesis that is consistent with the data is that both cell swelling and NEM cause inhibition of a protein kinase. Kinase inhibition causes net dephosphorylation of some key substrate (not necessarily the transport protein); dephosphorylation of this substrate, probably by type 1 protein phosphatase, causes transport activation.