title: The ATP and Mg2+ dependence of Na(+)-K(+)-2Cl- cotransport reflects a requirement for protein phosphorylation: studies using calyculin A.
abstract: 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/threonine phosphatase inhibitors 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 phosphatase-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 cAMP-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 (EC50 approximately 630 nM) and calcyulin A (EC50 approximately 8 nM) in stimulating the cotransporter indicate that a type-1 protein phosphatase is probably responsible for dephosphorylating the system. Cells stimulated by hypertonicity were also resistant to ATP or Mg2+ depletion suggesting that the mechanism of shrinkage-induced cotransport stimulation might also involve protein phosphatase modulation.
authors.affiliation: Department of Pharmacological and Physiological Sciences, University of Chicago, IL 60637.
authors.forename: H C
authors.initials: HC
authors.lastname: Palfrey
authors.forename: E B
authors.initials: EB
authors.lastname: Pewitt
mesh_terms: D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D001693:Biological Transport, Active / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D004372:Ducks; D004912:Erythrocytes / Q000378:metabolism; D006982:Hypertonic Solutions; D066298:In Vitro Techniques; D008274:Magnesium / Q000502:physiology*; D008387:Marine Toxins; D010080:Oxazoles / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters
chemical_list: D002352:Carrier Proteins; D002712:Chlorides; D006982:Hypertonic Solutions; D008387:Marine Toxins; D010080:Oxazoles; D010761:Phosphorus Radioisotopes; D028021:Sodium-Potassium-Chloride Symporters; C059041:calyculin A; D000255:Adenosine Triphosphate; D012964:Sodium; D000242:Cyclic AMP; D010749:Phosphoprotein Phosphatases; D008274:Magnesium; D011188:Potassium