Cloning, characterization, and chromosomal location of a novel human K+-Cl- cotransporter.
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 potential N-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 alpha, 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 of 86Rb 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.
Hiki
K
K
Department of Human Immunology, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science and University of Adelaide, Adelaide, South Australia 5000.
D'Andrea
R J
RJ
Furze
J
J
Crawford
J
J
Woollatt
E
E
Sutherland
G R
GR
Vadas
M A
MA
Gamble
J R
JR
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism*; D002884:Chromosomes, Human, Pair 15; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D004730:Endothelium, Vascular / Q000378:metabolism; D004831:Epilepsies, Myoclonic / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D011188:Potassium / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016415:Sequence Alignment; D027981:Symporters; D000096922:K Cl- Cotransporters
D002352:Carrier Proteins; D002712:Chlorides; D018076:DNA, Complementary; D011188:Potassium; D012333:RNA, Messenger; D027981:Symporters; D000096922:K Cl- Cotransporters