Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter

Jian Chao Xu, Christian Lytle, Tracey T. Zhu, John A Payne, Edward Benz, Bliss Forbush

Research output: Contribution to journalArticle

333 Citations (Scopus)

Abstract

By mediating the coupled movement of Na,K, and Cl ions across the plasma membrane of most animal cells, the bumetanide-sensitive Na-K-Cl cotransporter (NKCC) plays a vital role in the regulation of ionic balance and cell volume. The transporter is a central element in the process of vectorial salt transport in secretory and absorptive epithelia. A cDNA encoding a Na-K-Cl cotransport protein was isolated from a shark rectal gland library by screening with monoclonal antibodies to the native shark cotransporter. The 1191-residue protein predicted from the cDNA sequence has 12 putative transmembrane domains flanked by large cytoplasmic N and C termini. Regulatory phosphoacceptor residues in isolated peptides are identified as Thr-189 and Thr-1114 in the predicted sequence. Northern blot analysis identified a 7.4-kb mRNA in rectal gland and most other shark tissues; a 5.2- kb mRNA was restricted to shark kidney. Homology with an uncharacterized gene from Caenorhabditis elegans and with the thiazide-sensitive Na-Cl cotransporter of flounder urinary bladder was found over most of the coding region; shorter stretches of homology were found with a C. elegans cDNA and with an uncharacterized gene of cyanobacterium. Human HEK-293 cells have been stably transfected with the shark cDNA and shown to express Na-K-Cl cotransport activity with the bumetanide sensitivity of the shark protein. The expressed transporter is functionally quiescent in the host cells and can be activated by depleting the cells of chloride.

Original languageEnglish (US)
Pages (from-to)2201-2205
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number6
StatePublished - Mar 15 1994
Externally publishedYes

Fingerprint

Sodium-Potassium-Chloride Symporters
Bumetanide
Sharks
Molecular Cloning
Complementary DNA
Salt Gland
Caenorhabditis elegans
Flounder
Messenger RNA
Proteins
HEK293 Cells
Cyanobacteria
Cell Size
Northern Blotting
Genes
Chlorides
Urinary Bladder
Epithelium
Salts
Monoclonal Antibodies

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter. / Xu, Jian Chao; Lytle, Christian; Zhu, Tracey T.; Payne, John A; Benz, Edward; Forbush, Bliss.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 91, No. 6, 15.03.1994, p. 2201-2205.

Research output: Contribution to journalArticle

Xu, Jian Chao ; Lytle, Christian ; Zhu, Tracey T. ; Payne, John A ; Benz, Edward ; Forbush, Bliss. / Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter. In: Proceedings of the National Academy of Sciences of the United States of America. 1994 ; Vol. 91, No. 6. pp. 2201-2205.
@article{0c28477f417e4fcc97ed3b203b4ec913,
title = "Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter",
abstract = "By mediating the coupled movement of Na,K, and Cl ions across the plasma membrane of most animal cells, the bumetanide-sensitive Na-K-Cl cotransporter (NKCC) plays a vital role in the regulation of ionic balance and cell volume. The transporter is a central element in the process of vectorial salt transport in secretory and absorptive epithelia. A cDNA encoding a Na-K-Cl cotransport protein was isolated from a shark rectal gland library by screening with monoclonal antibodies to the native shark cotransporter. The 1191-residue protein predicted from the cDNA sequence has 12 putative transmembrane domains flanked by large cytoplasmic N and C termini. Regulatory phosphoacceptor residues in isolated peptides are identified as Thr-189 and Thr-1114 in the predicted sequence. Northern blot analysis identified a 7.4-kb mRNA in rectal gland and most other shark tissues; a 5.2- kb mRNA was restricted to shark kidney. Homology with an uncharacterized gene from Caenorhabditis elegans and with the thiazide-sensitive Na-Cl cotransporter of flounder urinary bladder was found over most of the coding region; shorter stretches of homology were found with a C. elegans cDNA and with an uncharacterized gene of cyanobacterium. Human HEK-293 cells have been stably transfected with the shark cDNA and shown to express Na-K-Cl cotransport activity with the bumetanide sensitivity of the shark protein. The expressed transporter is functionally quiescent in the host cells and can be activated by depleting the cells of chloride.",
author = "Xu, {Jian Chao} and Christian Lytle and Zhu, {Tracey T.} and Payne, {John A} and Edward Benz and Bliss Forbush",
year = "1994",
month = "3",
day = "15",
language = "English (US)",
volume = "91",
pages = "2201--2205",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "6",

}

TY - JOUR

T1 - Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter

AU - Xu, Jian Chao

AU - Lytle, Christian

AU - Zhu, Tracey T.

AU - Payne, John A

AU - Benz, Edward

AU - Forbush, Bliss

PY - 1994/3/15

Y1 - 1994/3/15

N2 - By mediating the coupled movement of Na,K, and Cl ions across the plasma membrane of most animal cells, the bumetanide-sensitive Na-K-Cl cotransporter (NKCC) plays a vital role in the regulation of ionic balance and cell volume. The transporter is a central element in the process of vectorial salt transport in secretory and absorptive epithelia. A cDNA encoding a Na-K-Cl cotransport protein was isolated from a shark rectal gland library by screening with monoclonal antibodies to the native shark cotransporter. The 1191-residue protein predicted from the cDNA sequence has 12 putative transmembrane domains flanked by large cytoplasmic N and C termini. Regulatory phosphoacceptor residues in isolated peptides are identified as Thr-189 and Thr-1114 in the predicted sequence. Northern blot analysis identified a 7.4-kb mRNA in rectal gland and most other shark tissues; a 5.2- kb mRNA was restricted to shark kidney. Homology with an uncharacterized gene from Caenorhabditis elegans and with the thiazide-sensitive Na-Cl cotransporter of flounder urinary bladder was found over most of the coding region; shorter stretches of homology were found with a C. elegans cDNA and with an uncharacterized gene of cyanobacterium. Human HEK-293 cells have been stably transfected with the shark cDNA and shown to express Na-K-Cl cotransport activity with the bumetanide sensitivity of the shark protein. The expressed transporter is functionally quiescent in the host cells and can be activated by depleting the cells of chloride.

AB - By mediating the coupled movement of Na,K, and Cl ions across the plasma membrane of most animal cells, the bumetanide-sensitive Na-K-Cl cotransporter (NKCC) plays a vital role in the regulation of ionic balance and cell volume. The transporter is a central element in the process of vectorial salt transport in secretory and absorptive epithelia. A cDNA encoding a Na-K-Cl cotransport protein was isolated from a shark rectal gland library by screening with monoclonal antibodies to the native shark cotransporter. The 1191-residue protein predicted from the cDNA sequence has 12 putative transmembrane domains flanked by large cytoplasmic N and C termini. Regulatory phosphoacceptor residues in isolated peptides are identified as Thr-189 and Thr-1114 in the predicted sequence. Northern blot analysis identified a 7.4-kb mRNA in rectal gland and most other shark tissues; a 5.2- kb mRNA was restricted to shark kidney. Homology with an uncharacterized gene from Caenorhabditis elegans and with the thiazide-sensitive Na-Cl cotransporter of flounder urinary bladder was found over most of the coding region; shorter stretches of homology were found with a C. elegans cDNA and with an uncharacterized gene of cyanobacterium. Human HEK-293 cells have been stably transfected with the shark cDNA and shown to express Na-K-Cl cotransport activity with the bumetanide sensitivity of the shark protein. The expressed transporter is functionally quiescent in the host cells and can be activated by depleting the cells of chloride.

UR - http://www.scopus.com/inward/record.url?scp=0028343934&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028343934&partnerID=8YFLogxK

M3 - Article

VL - 91

SP - 2201

EP - 2205

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 6

ER -