Role of protein phosphorylation in control of K flux pathways of trout red blood cells

Abstract

The role of protein phosphatases in the regulation of K flux pathways of the trout red blood cell has been investigated using the phosphatase inhibitors calyculin A and okadaic acid. Both inhibitors completely blocked an oxygenation-activated Cl-dependent K flux with a 50% inhibitory concentration of 17 and 675 nmol/l, respectively, but not the hypotonically activated Cl-independent K uptake. N-ethylmaleimide (NEM) and staurosporine caused an increase in the Cl-dependent flux. In both cases preincubation with calyculin A blocked activation but, when added during activation, it prevented any further increase with NEM but abolished the staurosporine-induced uptake. K uptake that was activated by NEM and "clamped" by calyculin A was volume sensitive, indicating a dual influence on this pathway. Chelerythrine, a protein kinase inhibitor, activated a Cl-independent K uptake that was unaffected by calyculin A. It is concluded that activation and deactivation of both Cl-dependent and Cl-independent pathways require changes in the phosphorylation of an as yet unidentified target protein(s), although with different sets of protein kinase and/or phosphatases. These observations also suggest a complex model of kinase-phosphatase regulation and provide drugs for the pharmacological definition and manipulation of Cl-dependent and Cl-independent K flux pathways.