1. Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human. A new member of the cation-chloride cotransporter family.
- Author
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Gillen CM, Brill S, Payne JA, and Forbush B 3rd
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Brain metabolism, Carrier Proteins metabolism, Cell Line, Chlorides metabolism, Cloning, Molecular, DNA Primers, DNA, Complementary, Databases, Factual, Humans, Kidney metabolism, Kinetics, Models, Structural, Molecular Sequence Data, Multigene Family, Open Reading Frames, Polymerase Chain Reaction, Potassium metabolism, Rabbits, Rats, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Rubidium metabolism, Sequence Homology, Amino Acid, Sequence Tagged Sites, Transfection, K Cl- Cotransporters, Carrier Proteins biosynthesis, Carrier Proteins chemistry, Phylogeny, Protein Structure, Secondary, Symporters
- Abstract
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.
- Published
- 1996
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