THE ATP AND MG2-K+-2CL- COTRANSPORT REFLECTS A REQUIREMENT FOR PROTEIN-PHOSPHORYLATION - STUDIES USING CALYCULIN-A( DEPENDENCE OF NA+)

Na+-K+-2Cl(-) cotransport activity has previously been shown to depend on both intracellular ATP and Mg2+, but the mechanisms remain unknown . Cotransport in avian erythrocytes can be stimulated by a variety of agents including cAMP and permeant serine/ hreonine phosphatase inhibi tors and is inhibited by prior depletion of either ATP with antimycin A, or Mg2+ by incubation in A23187 plus EDTA. However, when cells were first stimulated with cAMP rather than calyculin A then subjected to either depletion strategy, a differential effect was found. The phosph atase-inhibitor-treated cells were resistant to subsequent ATP or Mg2 depletion while cAMP-treated cells were sensitive to both treatments. Parallel examination of protein phosphorylation confirmed that ATP or Mg2+ depletion leads to dephosphorylation of membrane proteins in cAM P-treated but not in calyculin-A-treated cells. These results suggest that, for cotransport, ATP and Mg2+ are required primarily to maintain the system in a phosphorylated state rather than as direct modulators . The relative effectiveness of okadaic acid (EC(50) approximate to 63 0 nM) and calyculin A (EC(50) approximate to 8 nM) in stimulating the cotransporter indicate that a type-1 protein phosphatase is probably r esponsible for dephosphorylating the system. Cells stimulated by hyper tonicity were also resistant to ATP or Mg2+ depletion suggesting that the mechanism of shrinkage-induced cotransport stimulation might also involve protein phosphatase modulation.