We have studied the regulation of the K-Cl cotransporter KCC1 and its functional interaction with the Na-K-Cl cotransporter. K-Cl cotransporter activity was substantially activated in HEK-293 cells overexpressing KCC1 (KCC1-HEK) by hypotonic cell swelling, 50 mM external K, and pretreatment with N-ethylmaleimide (NEM). Bumetanide inhibited ⁸⁶Rb efflux in KCC1-HEK cells after cell swelling [inhibition constant (K_i) ~190 µM] and pretreatment with NEM (K_i ~60 µM). Thus regulation of KCC1 is consistent with properties of the red cell K-Cl cotransporter. To investigate functional interactions between K-Cl and Na-K-Cl cotransporters, we studied the relationship between Na-K-Cl cotransporter activation and intracellular Cl concentration ([Cl]_i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activity than controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells was activated <2-fold by low-Cl hypotonic prestimulation, compared with 10-fold activation in HEK-293 cells and >20-fold activation in cells overexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cells had lower resting [Cl]_i than HEK-293 cells; cell volume was not different among cell lines. We found a steep relationship between [Cl]_i and Na-K-Cl cotransport activity within the physiological range, supporting a primary role for [Cl]_i in activation of Na-K-Cl cotransport and in apical-basolateral cross talk in ion-transporting epithelia.

intracellular chloride; epithelial transport; cell volume

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