Regulatory phosphorylation of the secretory Na-K-Cl cotransporter: modulation by cytoplasmic Cl.
The effect of cytoplasmic Cl concentration ([Cl]i) on the activation state ([3H]benzmetanide binding rate) and phosphorylation state (32P incorporation) of the Na-K-Cl cotransporter was evaluated in secretory tubules isolated from the dogfish shark rectal gland. Reduction of [Cl]i at relatively constant cell volume (by removal of extracellular Cl or Na or by addition of bumetanide) increased cotransporter activation and phosphorylation. Raising extracellular K concentration ([K]o) from 4 to 80 mM, a maneuver that elevated [Cl]i above normal, reduced basal cotransport activity and rendered it entirely refractory to forskolin. High [K]o also blocked activation and phosphorylation in response to cell shrinkage, despite the fact that [Cl]i was already greatly elevated as a consequence of osmotic water loss. The phosphatase inhibitor calyculin A also promoted activation, but not in cells preexposed briefly to high [K]o. In summary, maneuvers than lower [Cl]i activate the cotransporter, whereas those that elevate [Cl]i (or prevent it from decreasing) block activation in response to secretory stimuli. Cell Cl appears to govern its own rate of entry via Na-K-Cl cotransport by impeding regulatory phosphorylation of the Na-K-Cl cotransport protein.
Lytle
C
C
Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672, USA.
Forbush
B
B
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism*; D007477:Ions; D008387:Marine Toxins; D009994:Osmolar Concentration; D010080:Oxazoles / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D012491:Salt Gland / Q000166:cytology / Q000378:metabolism; D012754:Sharks; D028021:Sodium-Potassium-Chloride Symporters; D013997:Time Factors
D002352:Carrier Proteins; D002712:Chlorides; D007477:Ions; D008387:Marine Toxins; D010080:Oxazoles; D028021:Sodium-Potassium-Chloride Symporters; C059041:calyculin A; D000242:Cyclic AMP; D010744:Phosphoric Monoester Hydrolases; D011188:Potassium