Regulation of Na+-K+-2Cl- cotransport by protein phosphorylation in ferret erythrocytes.

1. Na+-K+-2Cl- cotransport in ferret erythrocytes was measured as the bumetanide-sensitive uptake of 86Rb. 2. The resting cotransport rate was high but could be increased threefold by treating erythrocytes with calyculin A, a potent inhibitor of serine/threonine phosphatases. Twenty nanomolar was sufficient to maximally and rapidly (within 4 min) stimulate transport. 3. The effects of several kinase inhibitors were tested. High concentrations of K-252a, K-252b, calphostin C and hypericin caused less than 20 % inhibition. Staurosporine (IC50, 0.06 microM) and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1; IC50, 2.5 microM) were more potent but still only partially (40-50 %) inhibited transport, an effect mimicked by reducing ionized intracellular Mg2+ concentration to submicromolar levels. Genistein may inhibit all transport at a sufficiently high dose (IC50, 0.36 mM) perhaps by directly inhibiting the transporter. 4. Staurosporine, PP1 and the removal of Mg2+ all prevented subsequent stimulation by calyculin A, and all inhibited calyculin-stimulated transport by 20-30 %. The effects of staurosporine, PP1 and Mg2+ removal were not additive. 5. The phosphatase that dephosphorylates the cotransporter is probably Mg2+ (or possibly Ca2+ or Mn2+) sensitive and not the target for calyculin A. The data suggest that this phosphatase is inhibited by phosphorylation, and that it is the regulation of this process which is affected by calyculin A and the kinase inhibitors tested here. Phosphorylation of the phosphatase is probably regulated by members of the Src family of tyrosine kinases.