Cloning and characterization of the endothelial cell Na-K-Cl cotransporter

Martha E O'Donnell, T. R. Yerby, L. Gonsalves, James D Brandt, John A Payne

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Abstract

Purpose: Previous studies from this laboratory have shown that the Na-K-Cl cotransporter of endothelial cells plays a vital role in regulation of intracellular volume. We have also found that the Na-K-Cl cotransporter functions to regulate intracellular volume of trabecular meshwork (TM) cells. Recent studies have shown the existence of a family of cation-chloride cotransporters which includes the Na-K-Cl cotransporters of secretory and absorptive epithelial cells (NKCC1 and NKCC2, respectively). In order to clarify the function and regulation of endothelial and TM cotransporters at a molecular level, in the present study we cloned Na-K-Cl cotransporter cDNA from a bovine aortic endothelial (BAE) cell library. Methods: A 1.0 kb fragment of human NKCC1 DNA encoding the transmembrane region was used to screen a BAE cDNA library. Two clones that spanned the entire coding region were identified and sequenced. Results: The BAE cotransporter amino acid sequence was 86% identical to NKCC1, particularly in the transmembrane domains although significant variations were found at the N-terminus and in transmembrane segment 2. A survey of bovine tissue RNA using a BAE cotransporter cDNA probe revealed high expression of the cotransporter in brain, colon, stomach and kidney. In addition, the BAE cotransporter cDNA probe also identified the cotransporter in RNA of cultured bovine TM cells. A BAE cDNA fragment was cloned into an expression vector and was stably integrated into human embryonic kidney (HEK) cells to evaluate functional expression. Several lines exhibited markedly elevated cotransport activity compared to non-transfected HEK cells. Conclusions: These functional assays demonstrate that the cDNA cloned is indeed a member of the Na-K-Cl cotransporter family and is highly homologous with NKCC1 and further, that the TM cell cotransporter also exhibits homology with the endothelial cotransporter and NKCC1.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996

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Sodium-Potassium-Chloride Symporters
Organism Cloning
Endothelial Cells
Trabecular Meshwork
Complementary DNA
Kidney
Member 2 Solute Carrier Family 12
RNA
Gene Library
Cations
Chlorides
Amino Acid Sequence
Stomach
Colon
Clone Cells
Epithelial Cells

ASJC Scopus subject areas

  • Ophthalmology

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Cloning and characterization of the endothelial cell Na-K-Cl cotransporter. / O'Donnell, Martha E; Yerby, T. R.; Gonsalves, L.; Brandt, James D; Payne, John A.

In: Investigative Ophthalmology and Visual Science, Vol. 37, No. 3, 15.02.1996.

Research output: Contribution to journalArticle

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abstract = "Purpose: Previous studies from this laboratory have shown that the Na-K-Cl cotransporter of endothelial cells plays a vital role in regulation of intracellular volume. We have also found that the Na-K-Cl cotransporter functions to regulate intracellular volume of trabecular meshwork (TM) cells. Recent studies have shown the existence of a family of cation-chloride cotransporters which includes the Na-K-Cl cotransporters of secretory and absorptive epithelial cells (NKCC1 and NKCC2, respectively). In order to clarify the function and regulation of endothelial and TM cotransporters at a molecular level, in the present study we cloned Na-K-Cl cotransporter cDNA from a bovine aortic endothelial (BAE) cell library. Methods: A 1.0 kb fragment of human NKCC1 DNA encoding the transmembrane region was used to screen a BAE cDNA library. Two clones that spanned the entire coding region were identified and sequenced. Results: The BAE cotransporter amino acid sequence was 86{\%} identical to NKCC1, particularly in the transmembrane domains although significant variations were found at the N-terminus and in transmembrane segment 2. A survey of bovine tissue RNA using a BAE cotransporter cDNA probe revealed high expression of the cotransporter in brain, colon, stomach and kidney. In addition, the BAE cotransporter cDNA probe also identified the cotransporter in RNA of cultured bovine TM cells. A BAE cDNA fragment was cloned into an expression vector and was stably integrated into human embryonic kidney (HEK) cells to evaluate functional expression. Several lines exhibited markedly elevated cotransport activity compared to non-transfected HEK cells. Conclusions: These functional assays demonstrate that the cDNA cloned is indeed a member of the Na-K-Cl cotransporter family and is highly homologous with NKCC1 and further, that the TM cell cotransporter also exhibits homology with the endothelial cotransporter and NKCC1.",
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