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-3 2 incorporation) of the Na-K-Cl cotransporter was evaluated in secreto ry tubules isolated from the dogfish shark rectal gland. Reduction of [Cl](i) at relatively constant cell volume (by removal of extracellula r C1 or Na or by addition of bumetanide) increased cotransporter activ ation and phosphorylation. Raising extracellular K concentration ([K]( o)) from 4 to 80 mM, a maneuver that elevated [Cl](i) above normal, re duced basal cotransport activity and rendered it entirely refractory t o forskolin. High [K](o) also blocked activation and phosphorylation i n response to cell shrinkage, despite the fact that [Cl](i) was alread y greatly elevated as a consequence of osmotic water loss. The phospha tase inhibitor calyculin A also promoted activation, but not in cells preexposed briefly to high [K](o). In summary, maneuvers that lower [C l](i) activate the cotransporter, whereas those that elevate [Cl](i) ( or prevent it from decreasing) block activation in response to secreto ry stimuli. Cell Cl appears to govern its own rate of entry via Na-K-C l cotransport by impeding regulatory phosphorylation of the Na-K-Cl co transport protein.