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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 (Ki) approximately 190 microM] and pretreatment with NEM (Ki approximately 60 microM). 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.

We report the cloning, sequence analysis, tissue distribution, and functional expression of the K-Cl cotransport protein, KCC1. KCC1 was identified by searching the human expressed sequence tag data base, based on the expectation that it would be distantly related to the Na-K-Cl cotransporter. Rabbit KCC1 (rbKCC1) and rat KCC1 (rtKCC1) were cloned by screening rabbit kidney and rat brain cDNA libraries using homologous cDNA probes. Human KCC1 (hKCC1) was obtained from I.M.A.G.E. clones and in part by reverse transcription-polymerase chain reaction; it exhibits 97% identity with rbKCC1. KCC1 encodes a 1085-residue polypeptide with substantial sequence homology (24-25% identity) to the bumetanide-sensitive Na-K-Cl cotransporter (NKCC or BSC) and the thiazide-sensitive Na-Cl cotransporter (NCC or TSC). Hydropathy analysis of KCC1 indicates structural homology to NKCC, including 12 transmembrane domains, a large extracellular loop with potential N-linked glycosylation sites, and cytoplasmic N- and C-terminal regions. Northern blot analysis revealed a ubiquitously expressed 3. 8-kilobase transcript. Much of the genomic sequence of hKCC1 is in the data base, and the gene has been previously localized to 16q22.1 (Larsen, F., Solhein, J., Kristensen, T., Kolsto, A. B., and Prydz, H.(1993) Hum. Mol. Genet. 2, 1589-1595). Epitope-tagged rbKCC1 was stably expressed in human embryonic kidney (HEK 293) cells, resulting in production of a approximately150-kDa glycoprotein. The initial rate of 86Rb efflux from cells expressing rbKCC1 was more than 7 times greater than efflux from control cells and was inhibited by 2 mM furosemide; 86Rb efflux was stimulated by cell swelling. Uptake of 86Rb into rbKCC1 cells after a 15-min pretreatment with 1 mM N-ethylmaleimide was dependent on external chloride but not on external sodium, and was inhibited by furosemide with a Ki of approximately 40 microM and by bumetanide with a Ki of approximately 60 microM. These data demonstrate that the KCC1 cDNAs encode a widely expressed K-Cl cotransporter with the characteristics of the K-Cl transporter that has been characterized in red cells.