Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human: A new member of the cation-chloride cotransporter family

Christopher M. Gillen, Susan Brill, John A Payne, Bliss Forbush

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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 ~150-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 K(i) of ~40 μM and by bumetanide with a K(i) of ~60 μM. 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.

Original languageEnglish (US)
Pages (from-to)16237-16244
Number of pages8
JournalJournal of Biological Chemistry
Volume271
Issue number27
DOIs
StatePublished - 1996

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Sodium-Potassium-Chloride Symporters
Bumetanide
Cloning
Furosemide
Molecular Cloning
Member 4 Solute Carrier Family 12
Cations
Rats
Chlorides
Complementary DNA
Cells
Rabbits
Glycosylation
Ethylmaleimide
Polymerase chain reaction
Expressed Sequence Tags
Transcription
Gene Library
Swelling
Epitopes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular cloning and functional expression of the K-Cl cotransporter from rabbit, rat, and human : A new member of the cation-chloride cotransporter family. / Gillen, Christopher M.; Brill, Susan; Payne, John A; Forbush, Bliss.

In: Journal of Biological Chemistry, Vol. 271, No. 27, 1996, p. 16237-16244.

Research output: Contribution to journalArticle

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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 ~150-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 K(i) of ~40 μM and by bumetanide with a K(i) of ~60 μM. 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.",
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N2 - 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 ~150-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 K(i) of ~40 μM and by bumetanide with a K(i) of ~60 μM. 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.

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