Cloning, Characterization, and Chromosomal Location of a Novel Human K+-Cl− Cotransporter

doi:10.1074/jbc.274.15.10661

Cloning, Characterization, and Chromosomal Location of a Novel Human K⁺-Cl⁻ Cotransporter*

From the ^‡Department of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science and University of Adelaide, Adelaide, South Australia 5000 and ^‖Centre for Medical Genetics, Department of Cytogenetics and Molecular Genetics, Women’s and Children’s Hospital, North Adelaide, South Australia 5006, Australia

Abstract

Differential display polymerase chain reaction has been used to isolate genes regulated in vascular endothelial cells by the angiogenic factor vascular endothelial cell growth factor (VEGF). Analysis of one of the bands consistently up-regulated by VEGF led us to the identification of a cDNA from a human umbilical vein endothelial cell library that is 77% identical to the human K⁺-Cl⁻ cotransporter1 (KCC1). We have referred to the predicted protein as K⁺-Cl⁻cotransporter 3 (KCC3). Hydrophobicity analysis of the KCC3 amino acid sequence showed an almost identical pattern to KCC1, suggesting 12 membrane-spanning segments, a large extracellular loop with potentialN-glycosylation sites, and cytoplasmic N- and C-terminal regions. The KCC3 mRNA was highly expressed in brain, heart, skeletal muscle, and kidney, showing a distinct pattern and size from KCC1 and KCC2. The KCC3 mRNA level in endothelial cells increased on treatment with VEGF and decreased with the proinflammatory cytokine tumor necrosis factor α, whereas KCC1 mRNA levels remained unchanged. Stable overexpression of KCC3 cDNA in HEK293 cells produced a glycoprotein of approximately 150 kDa, which was reduced to 120 kDa by glycosidase digestion. An increased initial uptake rate of⁸⁶Rb was seen in clones with high KCC3 expression, which was dependent on extracellular Cl⁻ but not Na⁺and was inhibitable by the loop diuretic agent furosemide. The KCC3 genomic localization was shown to be 15q13 by fluorescence in situ hybridization. Radiation hybrid analysis placed KCC3 within an area associated with juvenile myoclonic epilepsy. These results suggest KCC3 is a new member of the KCC family that is under distinct regulation from KCC1.

Footnotes

↵* This work was supported by grants from National Health and Medical Research Council and the National Heart Foundation of Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Supported by Kyoto University and Ikadachi Hospital, Japan. Current address: Dept. of Pharmacology, Faculty of Medicine, Kyoto University, Yoshida Konoe Cho, Sakyo-ku, Kyoto 606 Japan.
↵¶ Supported by an HM Lloyd Senior Research Fellowship in Oncology from the University of Adelaide.
↵** These authors contributed equally to this paper.
↵‡ To whom correspondence should be addressed: Dept. of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science and University of Adelaide, Frome Rd., Adelaide, SA 5000, Australia. Tel.: 618--8222-3482; Fax: 618-8232-4092; E-mail:jennifer.gamble{at}imvs.sa.gov.au.
Abbreviations:

CCC

cation chloride cotransporter

NKCC

sodium potassium chloride cotransporter

KCC

potassium chloride cotransporter

VEGF

vascular endothelial cell growth factor

HUVEC

human umbilical vein endothelial cell

PCR

polymerase chain reaction

TNFα

tumor necrosis factor α

kb

kilobase(s)
- Received December 24, 1998.
- Revision received January 25, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.