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 ([H-3]benzmetanide binding rate) and phosphorylation state (P-32 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 C1 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 that 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.