Cloning and functional characterization of a cation-Cl- cotransporter-interacting protein.
To date, the cation-Cl(-) cotransporter (CCC) family comprises two branches of homologous membrane proteins. One branch includes the Na(+)-K(+)-Cl(-) cotransporters (NKCCs) and the Na(+)-Cl(-) cotransporter, and the other branch includes the K(+)-Cl(-) cotransporters. Here, we have isolated the first member of a third CCC family branch. This member shares approximately 25% identity in amino acid sequence with each of the other known mammalian CCCs. The corresponding cDNA, obtained from a human heart library and initially termed WO(3.3), encodes a 914-residue polypeptide of 96.2 kDa (calculated mass). Sequence analyses predict a 12-transmembrane domain (tm) region, two N-linked glycosylation sites between tm(5) and tm(6), and a large intracellular carboxyl terminus containing protein kinase C phosphorylation sites. Northern blot analysis uncovers an approximately 3.7-kilobase pair transcript present in muscle, placenta, brain, and kidney. With regard to function, WO(3. 3) expressed either in HEK-293 cells or Xenopus laevis oocytes does not increase Rb(+)-, Na(+)-, and Cl(-)-coupled transport during 5- or 6-h fluxes, respectively. In the oocyte, however, WO(3.3) specifically inhibits human NKCC1-mediated (86)Rb(+) flux. In addition, coimmunoprecipitation studies using lysates from WO(3. 3)-transfected HEK-293 cells suggest a direct interaction of WO(3.3) with endogenous NKCC. Thus, we have cloned and characterized the first putative heterologous CCC-interacting protein (CIP) known at present. CIP1 may be part of a novel family of proteins that modifies the activity or kinetics of CCCs through heterodimer formation.
Caron
L
L
Groupe de Recherche en Néphrologie, Department of Medicine, and the Unité de Recherche en Génétique Humaine et Moléculaire, Laval University, Québec G1R 2J6, Canada.
Rousseau
F
F
Gagnon
E
E
Isenring
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D002897:Chromosomes, Human, Pair 7 / Q000235:genetics; D003001:Cloning, Molecular; D019281:Dimerization; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D006031:Glycosylation; D006801:Humans; D017136:Ion Transport; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009865:Oocytes; D010766:Phosphorylation; D010802:Phylogeny; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism; D016415:Sequence Alignment; D012964:Sodium / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D002712:Chlorides; D012333:RNA, Messenger; C415467:SLC12A9 protein, human; D028021:Sodium-Potassium-Chloride Symporters; D012964:Sodium; D012413:Rubidium